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Understanding immune-modulatory efficacy in vitro

Boosting or suppressing our immune system represents an attractive adjunct in the treatment of infections including SARS-CoV-2, cancer, AIDS, malnutrition, age related problems and some inflammatory disorders. Thus, there has been a growing interest in exploring and developing novel drugs, natural or synthetic, that can manipulate our defence mechanism. Many of such studies, reported till date, have been designed to explore effect of the therapeutic on function of macrophages, being a key component in innate immune system. Indeed, RAW264.7, J774A.1, THP-1 and U937 cell lines act as ideal model systems for preliminary investigation and selection of dose for in vivo studies. Several bioassays have been standardized so far where many techniques require high throughput instruments, cost effective reagents and technical assistance that may hinder many scholars to perform a method demanding compilation of available protocols. In this review, we have taken an attempt for the first time to congregate commonly used in vitro immune-modulating techniques explaining their principles. The study detected that among about 40 different assays and more than 150 sets of primers, the methods of cell proliferation by MTT, phagocytosis by neutral red, NO detection by Griess reaction and estimation of expression of TLRs, COX-2, iNOS, TNF-α, IL-6 and IL-1β by PCR have been the most widely used to screen the therapeutics under investigation.


Introduction
In human, the immune system is equipped to fight off invading wide array of potential pathogens that may cause diseases. For the last 70 years, the health sector has heavily relied on antimicrobial drugs that has now resulted in generation of multiple-drug-resistant bacteria [1]. In that note, therapeutic strategies based on improving the defence mechanism have several advantages over the use of traditional antibiotics [2]. In fact, administration of immune boosters may circumvent the problem of rapid emergence of resistant variety. The therapeutics may also expand treatment options for immunocompromised patients such as people suffering from certain types of cancer, AIDS, malnutrition, chronic granulomatous disease or age-related problems as well as in the advent of a novel pathogen [3]. As of March 2020, the world is currently dealing with a global outbreak of Coronavirus disease 2019 (COVID- 19) caused by SARS-CoV-2, a new and different virus, and thus the existing vaccines are ineffective against it [4]. Scientists have suggested few approaches to counteract the infection where boosting the immune response in phase 1 has been found to be appropriate in addition to symptomatic treatment [5]. However, massive production of inflammatory cytokines by aberrant immune activation may in turn cause damage to host tissues. The situation emerges in the late stage of coronavirus disease where cytokine storm is a major reason for disease progression and eventual death [6]. Thus, administration of powerful anti-inflammatories that can suppress immune system along with antiviral drug could be effective in phase 2 [5]. The efficacy of nonsteroidal anti-inflammatory medications has also been documented in a number of clinical disorders, including rheumatoid arthritis, psoriasis, Crohn's disease, ulcerative colitis, gout, ankylosing spondylitis, dental pain, dysmenorrhea and headache [7].
On the whole, immunotherapy or biological therapy is the treatment of diseases, designed either to amplify an immune response, called immune-stimulation or reduce the response, called suppression immunotherapies [8]. Currently, there is a greater interest in natural compounds, such as dietary supplement and herbal remedies, which have been used for centuries as immune-modulators. Marine sources with diverse constituents (sulphated polysaccharide, terpenes, cyclic depsipeptide, polysaccharides, bioglycan, polyhydroxylated lactone, macrocyclic lactones, cyclic tripeptide) and medicinal plants with various active components (flavonoids, curcumin, saponin, carotenoids, alkaloid, tannic acid, tocopherol, polyphenol, ascorbate, polysaccharides) have proved their innate immune boosting abilities [8,9]. Fungi have also emerged as potential source of body defence enhancers where polysaccharides (in particular β-D-glucans), polysaccharopeptides and proteins have been recognized as bioactive compounds. Indeed, several mushroom derived macromolecules namely Schizophyllan, Lentinan, Krestin and polysaccharide-protein complex are now available as biological response modifiers in market [10]. On the other hand, alkaloids, glycosides, terpenoids, polyphenols, resins, essential oil, flavonoids, phenolic compounds, cannabinoids, steroids, fatty acids, lignans and glycoproteins isolated from various bio-resources have shown significant anti-inflammatory activities [11]. Numerous metal and metal oxide nanoparticles such as gold, silver, titanium dioxide, zinc oxide and selenium have also demonstrated to possess the medicinal prospect [12]. Most of these ingredients have been evaluated using in vitro system where macrophage cell lines are popularly used as an experimental model [13]. The monocytes can effortlessly be maintained; as a result, they are gradually being recognized as an ideal system for studies of immunomodulation and immunomechanics [14].
To date, a variety of in vitro immune modulating assays have been standardized to determine effect of a drug on specific function of macrophages, as published in our previous articles [15][16][17][18][19][20]. One-dimension approach can provide unequivocal result; thus, performing various methods is the best solution to reach the conclusion. However, most of these techniques are associated with instrumental, technical and practical limitations hindering researchers to perform an assay. It is thus important to have a complete knowledge about alternative methods targeting the same output. Therefore, a compiled description of all available in vitro models is an extreme requirement. In this regard, we intended to congregate the most commonly performed assays, suitable to estimate either immune-stimulatory or anti-inflammatory potency, as they execute opposite responses of the same method ( Supplementary  Fig. 1). The present review is expected to be highly beneficial not only to all immunologists to design new methods but also to pharmacists and biologists to develop novel drugs by selecting convenient assays.

Detection of lipopolysaccharide (LPS) contamination
The major contaminant found in biological substances is endotoxin, also called LPS, derived from cell membrane of Gram negative bacteria. Lipopolysaccharide is known to activate the immune system specifically macrophages with release of different pro-inflammatory mediators; as a result, LPS (generally at 5 μg/ml concentration) is used as a positive control in all immune-stimulation assays [17,21]. Thus, it is essential to detect presence of endotoxin in pharmaceutical products to understand sole effect of the drug. The commonly followed technique in this context is Limulus Amoebocyte Lysate (LAL) assay based on clotting ability of Limulus polyphemus blood upon exposure to endotoxin [22]. Briefly, 100 μl of test sample (10 mg/ml) or standard (positive control) or endotoxin-free water (negative control) are mixed with 100 μl of LAL for 1 h at 37 • C [23]. Gel is formed in proportional to endotoxin sensitivity Table 1 Overview of some commonly used cell lines in immunomodulatory research [13,[29][30][31]. The publication numbers were derived by searching in PubMed (https:// www.ncbi.nlm.nih.gov/pubmed/) on November 18, 2021 using the following criteria: (i) RAW 264.7 AND immune activation; (ii) RAW 264.7 AND anti inflammation; (iii) J774 AND immune activation; (iv) J774 AND anti inflammation; (v) THP-1 AND immune activation; (vi) THP-1 AND anti inflammation; (vii) U937 AND immune activation; (viii) U937 AND anti inflammation; (vi) THP-1 AND anti inflammation; (ix) HL-60 AND immune activation; (x) HL-60 AND anti inflammation. and 0.5 endotoxin unit (EU)/ml concentration is considered as the threshold level [22].

Cell culture
Murine macrophage cell lines such as RAW264.7 and J774A.1 are well-established typical systems in immunology and cell biology for their multi-tasking aptitude (Table 1). J774A.1 cell line was established from a female BALB/c/NIH mouse with a reticulum cell sarcoma [24]. These cells are cultured in Dulbecco's Modified Eagle's Medium (DMEM) or Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% fovin serum albumin (FBS), 100 μg/ml streptomycin and 100 units/ml penicillin at 37 • C in 5% CO 2 humidified atmosphere cultivator. The same condition can be used for culturing RAW264.7 macrophage cells. The cell line was constituted by SW Russell from ascites of a male BALB/c mouse bearing a tumor induced by Abelson murine leukemia virus [13]. Generally, the cells are cultivated for 36-48 h to reach the logarithmic phase and then used for experiments. Another option could be THP-1 (human acute monocytic leukemia derived cell line) or U937 (human myelomonocytic tumor), which differentiate into macrophages after exposure to phorbol-12-myristate-13-acetate (PMA) or a combination of PMA and LPS [25]. Next to these cells is HL-60 (human myelogenous leukemic cell line) that can be induced to differentiate in vitro into monocyte/macrophage-like cells, granulocyte-like cells and eosinophils after treatment with definite chemicals. As such, the cells are known to be differentiated into maturing macrophages in exposure with various differentiation-inducing agents such as 1,25-dihydroxyvitamin D 3 , phorbol esters and sodium butyrate. While, dimethyl sulfoxide (DMSO) or all trans retinoic acid treatment induces HL-60 cells to differentiate to neutrophil-like cell [26]. Another less commonly used cell line is PLB-985, established in 1985, which was later identified as a sub-line of HL-60 and thus exhibit similar properties [27]. Comparatively, most of the experimentations have been performed using murine monocytes which might be due to fast growth rate and less variability of macrophages from Mus musculusis than that of Homo sapiens [28].

Determination of cell density by trypan blue dye exclusion test
Confluency of cells in culture causes slowdown of metabolism, decrease in cell division and contact inhibition of growth. As a result, the condition may lead to loss of linearity between cell number and effect of drug. So, starting an investigation with definite number of viable cells is vital to perform successful experiments. In this context, trypan blue dye exclusion test is the most commonly utilized assay to distinguish live and dead cells selectively [32]. This negatively charged diazo stain interacts with cells only when the membrane is disrupted. Indeed, the dye is capable to penetrate the cytoplasm of dead cells only. As a result, these cells appear with distinctive blue colour when observed under a microscope. In contrast, live cells with intact membrane exclude such stain allowing discrimination with non-viable cells [33]. However, the process cannot be used to differentiate healthy cells and cells that are alive but losing functions. To perform the assay, 0.4% (w/v) trypan blue solution is prepared in phosphate buffered solution or PBS (pH 7.2). In a microtube, 20 μl of the solution is mixed with 20 μl of cell suspension and 10 μl is loaded onto a haemocytometer. Viable and non-viable cells, represented as transparent and blue colour respectively, are counted following standard manner within 5 min of mixing [34]. For healthy log-phase culture, cell viability should be at least 95% and these cells can be used for further experimentation.

Cytotoxicity/cell proliferation assay
Cell viability and cytotoxicity studies are valuable tools for sensitivity testing of chemicals that also provide preliminary information for in vivo studies. A variety of methods have been developed so far to determine proliferation in mammalian cells. These techniques are established on measurement of cellular DNA content, direct counting of viable cells by trypan blue dye or estimation of metabolic activity [35]. The latter procedure is based on mitochondrial dehydrogenase activity and hence is reliable, accurate, easy-to-use, safe and far superior to other assays. In this note, a range of stains have been developed that produce a specific colour (Fig. 1). Generally, the monocytes are cultured at a density of 1 × 10 4 cells/well in 96-well plate overnight and then treated with stimulus. Following incubation, substrate is added and the ultimate colour intensity is measured by enzyme-linked immune sorbent assay (ELISA) plate reader. The cell viability can be calculated by using following equation: Where A is optical density (OD) of the treated cells; B is OD of control wells (culture medium without cells); and C is OD of negative control (culture medium with cells). Non-cytotoxic concentrations of sample are selected to conduct downstream immune-stimulating assessments.

MTT
The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay was first described in 1983 and gradually has been modified by different authors. The method is based on reduction of yellow coloured water soluble MTT by mitochondrial succinate dehydrogenase activity in living cells into insoluble dark blue or purple formazan product [35][36][37][38]. The technique thus has great applicability for detection of metabolically active cells; even if they are not dividing. However, the disadvantage of the method is that the final product, formazan crystals injure cells and requires an organic solvent (DMSO or HCl/isopropanol) for solubilisation [38]. To perform the assay, 20 μl from MTT solution (5 mg/ml prepared in media or PBS) is added to each well to make final concentration of 0.5 mg/ml. After 4 h incubation at 37 • C, the supernatant is discarded and 150 μl DMSO is added. The plate is incubated for 10 min with frequent shaking to ensure complete solubilisation and absorbance is measured at 570 nm [39]. Else, 50 μl MTT stop solution (10% SDS in 10 mM HCl) can be added instead of DMSO followed by incubation for 24 h and measurement of absorbance at 540 nm [40].

MTS
MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), also called as Owen's reagent, is a weakly acidic inner salt closely related to MTT. This less toxic component is a negatively charged compound that cannot readily penetrate cells. So, the reagent is applied in combination with electron acceptor either phenazine methyl sulfate (PMS) or phenazine ethyl sulfate (PES). These intermediates can enter cell surface and exit cells to convert MTS to formazan product which is highly soluble in culture media. This transformation is thought to be accomplished by NADP(H)-dependent dehydrogenase enzyme in metabolically active cells [38]. Generally, 20 μl of MTS containing PES reagent is added in each well at the final concentration of 0.33 mg/ml. After 2 h incubation, the absorbance is determined at 490-500 nm using microplate reader [41].

WST
Recently, a new generation of water soluble tetrazolium salts has been developed of which WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) is the prototype [42]. WST-1 is a negatively charged disulfonated inner salt containing an iodine residue that yields a highly water soluble formazan extracellularly in presence of NADH and an electron mediator such as 1-methoxy PMS [43]. With the advantage over conventional tetrazolium salts that both WST-1 and its formazan are very stable in solution and they have extremely low cytotoxicity. Till date, several other tetrazolium salts have been developed in WST series, perhaps the most useful being WST-8 and is being marketed independently as Cell Counting Kit-8 (CCK-8). Since, WST-8, formazan product and mPMS have no cytotoxicity in cell culture media, additional experiments may be carried out using the same cells from the previous assay [38]. In general, 20 μl of WST-1 or 10 μl in case of CCK-8 solution is added to 200 μl reaction mixture in each well and optical density is measured at 450 nm after 4 h incubation at 37 • C [44].

XTT
XTT (sodium 3-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4methoxy-6-nitro) benzene sulfonic acid hydrate) cell proliferation assay was first described in 1988 as an effective method to measure cell growth and drug sensitivity in tumour cell lines. The compound contains two sulfonate groups giving them a net negative charge that allows excluding them from cells. Thus, XTT dye reduction occurs at the cell surface facilitated by trans-plasma membrane electron transport and the colourless or slightly yellow compound is converted to brightly orange, soluble formazan product. This colour change is accomplished by breaking apart the positively charged quaternary tetarzole ring [45]. To perform the assay, 50 μl of XTT solution is added to each well for 10 h at 37 • C in 5% CO 2 incubator and the optical density is measured at 490 nm [46].

Alamar blue
Resazurin (7-hydroxy-10-oxido-phenoxazin-10-ium-3-one) is a redox dye that has been applied to assess microbial contamination in milk and biological fluids since 1950s [45]. The assay is based on entry of resazurin (non-fluorescent blue coloured) into cytosol where it accepts electron from NADH, NADPH, FMNH, FADH and cytochromes. As a result, the dye is reduced to red coloured, highly fluorescent compound namely resorufin. The result can be depicted using a fluorescence detector with 545/15 nm excitation and 580/10 nm emission wavelength [37]. Major advantage of the method is non-toxicity to cells and high stability in culture media allowing continue monitoring of cultures over time. Consequently, the dye became commercially available since 1993 in the name of AlamarBlue or vibrant or UptiBlue. At first, Resazurin solution is prepared by dissolving the reagent in PBS (0.001% w/v) or in DMEM (10%) in dark and sterile filtered (0.22 μm). Then, the treated media is aspirated, replaced with 100 μl of resazurin solution in each well and resultant fluorescence is measured after 1-2 h incubation [47,48].

CellTiter-Glo
CellTiter-Glo utilizes the ability of metabolically active cells to catalyse luciferase reaction by its own ATP, yielding a measurable product. The reagent has ability to lysis cell membranes to release ATP and inhibit endogenous ATPases so that no further ATP is synthesized. The chemical also provides luciferin and luciferase to initiate bioluminescent reaction with the help of ATP discharged from cells; whereby intensity of final product directly signifies the number of living cells [36]. To perform the assay, cells are allowed to equilibrate for 30 min at room temperature (RT) after treatment. Then, substrate is added and samples are analysed with a Fluoroscan [49].

Crystal violet
The technique was initially established for cell number measurement in monolayer cultures. It is a simple, non-enzymatic assay for quick analysis of quantity of viable adherent cells and colonies. The assay takes advantage of affinity between dye and external surface of DNA double helix [36]. While, disadvantage of the assay is that if all dead cells are not removed from well before staining, they will be regarded as viable cells because of non-specific staining by crystal violet [37]. The generalised protocol is that, macrophages are washed with PBS and incubated with 0.2% (w/v) crystal violet containing 2% (v/v) ethanol in PBS at 37 • C for 30 min. Finally, macrophages are washed with PBS and the crystal violet is solubilized with 33% (v/v) acetic acid in water. Absorbance at 540 nm is measured in a microplate reader [50].

Cell cycle analysis
Macrophage cells (1 × 10 6 cells/ml) are seeded in 6-well plates and exposed to stimuli. After treatment, cells are washed in PBS and collected. They are then fixed in 70% glacial ethanol, washed in PBS, resuspended in 1 ml of PBS containing 50 U/ml RNase and 50 μg/ml propidium iodide (PI) and then incubated in dark for 40 min at 4 • C. Cell cycle analysis is performed by flow cytometry and the population of cells in various phases are calculated [51].

Morphological observation
After encountering stimuli, macrophages are induced to undergo morphodynamics such as increase in cell size, production of thin sheets of cell edges called filopodia (contains actin filaments) or lamellopodia (bears actin spikes) [21]. Change in morphological contour hence represents a clear indication of macrophage functionality activation or repression.

Bright field light microscopy
Cells are seeded on round glass coverslips at a density of 2 × 10 5 cells/well in 24-well plate. Following treatment with a stimulus, the supernatant is discarded and replaced with 4% paraformaldehyde fixative (300 μl/well). The plate is incubated at 8 • C for 2 h, wells are washed thoroughly with PBS (300 μl/well) to remove fixative and adherent cells are serially dehydrated in an ethanol series at 70, 80, 90 and 95% (5 min each bath). For cell staining, Harris hematoxylin (300 μl/well) is added for 1 min and the excess stain is removed by washing the coverslips with distilled water. Afterwards, 300 μl/well aqueous eosin is added for 30 s and rinsed. The glass coverslips are allowed to dry, mounted with Entellan on clean slide and analysed under bright field microscope [52].

Fluorescence microscopy
Macrophage cells are cultured in presence of sample on cover slides in 12-well plate or directly in 6-well plate for definite period of time. After incubation, cells are washed with PBS and incubated with DAPI (4 ′ ,6-diamidino-2-phenylindole) (1 μg/ml in PBS) for 15 min [20,21,53]. The cells may also be stained with DiOC 6 (3,3 ′ -dihexyloxacarbocyanine iodide) along with DAPI as shown in Supplementary  Fig. 2. Alternatively, cells are fixed in 4% (v/v) paraformaldehyde for 15 min and stained with DAPI (300 nmol/l) for 30 min at RT. Then cellular and nuclear morphology can be visualized, examined, and photographed by fluorescence microscopy [54].

Scanning electron microscopy (SEM) or transmission electron microscopy (TEM)
Treated and untreated macrophages are cultured on 6-well plate or glass coverslips in 24-well plates. After incubation, cells are washed with PBS, fixed overnight with 2-2.5% glutaraldehyde in 0.1 M cacodylate buffer and followed by post fixation with 1% osmium tetroxide for 2 h. Subsequently, the fixed cells are dehydrated through a graded series of alcohol and dried under a Critical Point Dryer. Further the cells are sputter-coated with gold. The morphological changes can be observed under an electron microscope and photographed [55].

Determination of phagocytic uptake
The term phagocytosis is used to define the cellular engulfment of particles larger than 0.5 μm in diameter. In immune response, the first and imperative defence function of macrophages is represented by phagocytosis that causes ingestion as well as elimination of pathogenic or inflammatory particles [56]. At first, antigens are recognized directly by pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptor (PRR) of innate immune system or indirectly by a process called opsonisation [57]. After PRR engagement, the foreign particles are internalized via protrusions of cell membrane forming a vesicle called phagosome. These phagosomes are transported within cytosol and its content becomes gradually acidified. After complete maturation, phagosome fuses with lysosome that bears a range of hydrolytic enzymes and anti-microbial peptides. This highly oxidative and acidic environment causes oxidative burst and subsequent elimination of its contents by exocytosis [58]. Thus, increase in phagocytic activity can definitely help to boost immunity and also signals for macrophage stimulation. Currently, there are a number of methods available to assess in vitro phagocytic activity. Most of these techniques use latex minibeads (0.75 mm), zymosan, Escherichia coli or yeast particles and some of these components are available in market labelled with fluorescein isothiocyanate (FITC).

Zymosan particles
Zymosan is an insoluble β-1,3-glucan polysaccharide, extracted from Saccharomyces cerevisiae and has been widely used for more than 50 years as a perfect model for microbial particle in studies on immune response. Since the preparation contains PAMP, they are easily recognized by innate immune recognition receptors stimulating phagocytosis and inflammatory cytokine production [59]. To perform the assay, macrophages are cultured in 24-well plate containing glass coverslips (13 mm diameter). After treatment with sample, they are allowed to phagocytose zymosan (1 × 10 6 particles/well) for 1 h at 37 • C. The cells are washed with PBS to remove non-digested particles, coverslips are fixed with 4% paraformaldehyde, stained with May-Grünwald reagent or Wright's stain [50]. The engulfment power can be investigated microscopically by counting individual cells and calculating average number of particles internalized per 100/200 macrophages for each experimental treatment. Phagocytic index (PI) is used to express overall effect and determined by using the following formula [60]: PI = Percentage phagocytosis × mean number of particles per cell

Killed yeast cells
Commercial baker's yeast (10-30 mg) is mixed in 10 ml PBS and heat-killed (80 • C for 15 min). Hundred μl of yeast cell suspension (around 1 × 10 8 cells/ml) are flooded onto macrophage cells on slide or 24-well plate. After incubation at 37 • C for 1.5-2 h, the cells are washed thrice with PBS to remove non-phagocytosed cells and air dried. Finally, macrophages are fixed in 100% methanol, stained with Giemsa dye or 0.1% crystal violet for 3-5 min (Supplementary Fig. 3). Cells are dried and counted without any predetermined pattern to calculate phagocytosis percentage and phagocytic index [61].

Live bacterial cells
After treatment, supernatant is removed and macrophage cells, cultured in 24-well plate, are flooded with 100 μl of freshly prepared E. coli cell suspension (2 × 10 6 cells/ml) and incubated at 37 • C. After 90 min, each well is washed to remove non-phagocytosed bacterial cells. Then cells can be lysed with 0.1% SDS and the lysates are plated on agar. After overnight incubation at 37 • C, colonies are counted [61].

FITC labelled particles
Cells are cultured in 6-well plate or 35 mm dish or on glass coverslips in 24-well plate. After incubation, treated medium is replaced with 1 ml PBS containing 100 μl FITC labelled beads (diameter: 2 μm) and incubated at 37 • C for 30 min to 2 h in dark. Finally, the adherent cells are washed thrice with PBS, fixed with paraformaldehyde (4% in PBS) for 30 min or 100 μl of 10% Triton X-100 and finally excess beads are removed by washing. Phagocytosis can also be stopped by repeated washing with cold PBS. Otherwise, the cells can be centrifuged for 5 min at 400×g at RT. The supernatant is removed and the cells are washed two times with 1 ml of PBS. Finally, cells are suspended in 500 μl of PBS and immediately analysed by flow cytometer [62].

Determination of pinocytic activity by neutral red assay
Pinocytosis is another type of endocytotic process by which macrophages take up fluid from surrounding medium along with molecules contained in it. To detect the potentiality, macrophages are seeded in 96well plate at 2 × 10 4 cells/well and after 6 h, incubated with stimuli.
Following treatment, 100 μl of 0.075%-0.1% neutral red prepared in 10 mM PBS or culture medium is added in each well. The reaction is incubated for one-two h followed by washing cells by PBS for three times to remove residual natural red solution. Then, 100-200 μl of cell lysis buffer (1:1 of ethanol:acetic acid v/v or ethanol: 1 mol/l acetic acid = 1:1 or 1% glacial acetic acid:ethanol = 1:1 or 1% acetic acid solution: 50% ethanol = 1:1) is added in each well and the mixture is incubated at RT for 4 h or overnight. Absorbance is read at 540 nm using ELISA plate reader. Pinocytic activity is expressed as pinocytic index and calculated as described in NBT assay at the phagocytosis section [20,56,63].

Intracellular calcium assay
Calcium ion is a ubiquitous second messenger that controls multiple processes in immune cells, including chemotaxis, adhesion, and secretion of pro-and anti-inflammatory cytokines. Resting cells have a Ca 2+ concentration around 100 nM which may quickly reach up to 1 μM when cells are stimulated [38]. Release of Ca 2+ from endoplasmic reticulum (ER) activates store-operated Ca 2+ entry channels in plasma and/or phagosomal membrane, leading to elevations in cytosolic Ca 2+ concentration. The phenomenon helps in efficient ingestion of foreign particles by some phagocytic receptors, controls subsequent steps involved in maturation of phagosome, solubilisation of surrounding actin meshwork, fusion of phagosomes with granules containing lytic enzymes and activation of superoxide generating NADPH oxidase complex [64]. This elevated level of cytosolic calcium, in turn, triggers transcription factors (TFs) such as activator protein-1 (AP-1; a heterodimeric protein constituted of c-Fos and c-Jun) and signal transducer and activator of transcription (STAT)1, STAT3; subsequently increasing transcription of pro-inflammatory target genes [65]. The change in intracellular Ca 2+ concentration signifies activation state of macrophages which can be detected by using certain stains.

Fluo-4 dye
Fluo-4 (C 36 H 30 F 2 N 2 O 13 ) is a dynamic single-wavelength fluorescent Ca 2+ indicator and an increase in fluorescence intensity reflects a rise in the cytoplasmic Ca 2+ level [66]. To execute the technique, macrophage cells are seeded in 96-well plates and after treatment the media is removed. Then, cells are incubated with 100 μl of dye loading solution for 30 min and fluorescence intensity is determined spectrofluorometrically with excitation and emission filters of 485 nm and 535 nm respectively [65].

Fura-2 dye
Fura-2 (C 44 H 47 N 3 O 24 ) represents another fluorescent Ca 2+ indicator that is widely used to monitor cytoplasmic Ca 2+ levels. Upon Ca 2+ binding, the fluorescence excitation spectrum is shifted toward shorter wavelength. Usually, the intensity of fluorescence induced by 340 nm and 380 nm excitation and emitted at 510 nm is measured, and therefore Fura-2 is a ratiometric dye that minimizes effects of photobleaching, leakage and uneven loading. The ratio of F340/F380 fluorescence intensity is proportional to the cytoplasmic Ca 2+ level [66]. The generalised protocol is that, after treatment macrophages are incubated with 5 μM of Fura-2 for 45 min at 37 • C. They are then washed twice with Hank's Balanced Salt Solution (HBSS) buffer (pH 7.2). Ratiometric calcium imaging is performed with an inverted fluorescence microscope [67].

Measurement of NO production
Nitric oxide is a membrane-permeable inorganic gas as well as free radical. It is synthesized from L-arginine by nitric oxide synthase and released by macrophages in response to pathogens. Therefore, increase in NO production is considered as an indication of macrophage activation [68]. However, this gaseous molecule is extremely short lived and rapidly oxidized to nitrate and/or nitrite by oxygen. Thus, measurement of stable and non-volatile breakdown product of NO like nitrite (NO 2 − ) is the best-known mean to investigate NO formation [69].

Griess reagent (iNtRON)
In Griess reaction, established in 1879, NO 2 − is first treated with sulphanilamide, a diazotizing agent, in acidic environment to form a diazonium salt. This transient intermediate is then allowed to react with N-[1-napnthyl]-ethylenediamide dihydrochloride (NED) to generate purple coloured stable azo compound [69]. To perform the assay, macrophage cells in 96-well plates (1 × 10 5 cells/well) or 24-well plates (3 × 10 5 cells/well) are pre-incubated for 12 h. Following treatment, 100 μl of Griess reagent (1% w/v sulphanilamide, 0.1% w/v NED in 5% phosphoric acid) is mixed with 100 μl of culture supernatant. The mixture is incubated at RT for 15 min and optical density is measured spectrophotometrically at 540 nm. The nitrite concentration is calculated from a standard curve derived from a reaction with sodium nitrite (0-100 μM) [40].

DAF2-DA
The assay is a fluorescence-based method that uses a cell permanent 4,5-diaminofluorescein diacetate (DAF2-DA) as NO detector. Diacetate (DA) part of the dye is hydrolysed in presence of cellular esterases to produce DAF2 compound that reacts with NO to produce a fluorescent triazolofluorescein (DAF-2T). To follow the assay, cells are seeded on coverslips in 6-well plates overnight and then treated with sample for desired time. Thereafter, cells are stained with 10 μM/ml DAF2-DA for 20-30 min at RT, washed with PBS and then fluorescence intensity is recorded at ex/em 480/535 nm [70].

Analysis of ROS production
The term ROS describes a group of molecules with at least one oxygen atom and with higher reactivity than O 2 [71]. They play an important role for antimicrobial activity of macrophages. In fact after pathogen recognition and phagocytosis, cytosolic subunits of NADPH oxidase (NOX) are phosphorylated and migrate to the phagosome membrane to form a functional enzyme with membrane-bound subunits. Following NADPH oxidase assembling, formation of superoxide radical (O 2 •− ) takes place inside the phagosome for up to 2 h approximately and the phenomenon is called oxidative burst. Eventually NO is produced in the cytosol of macrophages and readily diffuses across phagosome membrane due to its hydrophobic nature [72]. Nitric oxide then reacts with superoxide anion resulting in reactive nitrogen species (RNS) including peroxynitrite (ONOO − ). These oxidants induce nitrosative stress that promotes ROS production and are capable to kill microorganisms [73]. Release of ROS by the inflammatory cells can be quantified by various methods such as electron spin resonance spectroscopy, spectrophotometric measurements and chemiluminescence.

L-012
L-012, (8-amino-5-chloro-7-phenylpyrido [3,4-d]pyridazine-1,4 (2H,3H)dione) is a luminol-derivative chemiluminescence probe and highly sensitive for ROS. To carry out an assay for ROS detection, culture supernatant of treated and untreated macrophage cells is removed and replaced by an equal volume of HBSS supplemented with 25 μM L-012 and 5 μg/ml horseradish peroxidase (HRP) with or without PMA. Reactions are monitored on a Fluoroscan microtiter plate reader at 37 • C. Chemiluminescence is measured every 2 min for 2 h and is expressed as the integrated response over the time [49].

DCHFDA
The fluorogenic probe 2 ′ ,7 ′ -dichlorofluorescein-diacetate (DCHFDA) is able to diffuse into cell and deacetylated by cellular esterase. The resulting product is a non-fluorescent compound which is then oxidized by ROS into 2 ′ ,7 ′ -dichlorofluorescein (DCF). The resulting compound is highly fluorescent; hence can be detected by fluorescence spectroscopy [18]. To perform the assay, cells are washed with DMEM after treatment and stained with 10 μM of DCFDA at 37 • C for 20 min. Cells are washed with PBS twice and the fluorescence intensities can be measured at 485/20 nm excitation wavelength and 528/20 nm emission wavelength using a multimode microplate reader [74] or flow cytometry [20].

CM-DCFH
Intracellular ROS can also be measured by detecting the fluorescent intensity of carboxyl-2 ′ ,7 ′ -dichlorofluorescein diacetate (CM-DCFH) oxidized product, CM-DCF. After treatment, cells are harvested and washed with cold PBS. Washed cells are further incubated with 10 mM CM-DCFH at RT for 30 min. Relative fluorescent intensity of the fluorophore can be detected at ex/em 485/530 nm or using a fluorescence microscope [75].

Dihydrorhodamine123
Dihydrorhodamine 123 is a nonfluorescent and uncharged ROS indicator that can diffuse passively across membranes. The molecule in presence of ROS is oxidized to cationic rhodamine 123 which is trapped in mitochondria and exhibits green fluorescence [76]. The dihydro-rhodamine123 is added to each well (10 μM/well) containing treated or untreated macrophage cells and incubated for 15-30 min at RT. The fluorescent intensity is analysed by spectrofluorometer with excitation and emission wavelengths of 485/15 and 535/10 nm respectively [65].

Reduction of NBT
Macrophage cells (2 × 10 5 cells/ml) are plated on 96-well plates and cultured with sample. The cells are further incubated with 200 μl of PBS containing 0.1% of nitro-blue tetrazolium (NBT) and 2 μg/ml PMA at 37 • C for 30 min. This membrane permeable, water-soluble, yellowcoloured NBT reacts with superoxide anion produced inside phagocytic cells and is reduced to blue coloured formazan. The reaction is terminated with 80 μl of 70% methanol and cells are dried. The blue formazan produced is then dissolved in 2 M KOH (120 μl) and DMSO (140 μl) and absorbance is measured by a spectrophotometer at 620/10 nm against KOH/DMSO [77].

Measurement of H 2 O 2 production
Hydrogen peroxide (H 2 O 2 ), a primary component of ROS, is an uncharged, stable and freely diffusible second messenger. The agent is commonly produced from superoxide by superoxide dismutase (SOD) 1 and 2 during oxidative stress generated in phagocytic cells [73]. While superoxide crosses cell membrane poorly, H 2 O 2 diffuses easily between extra-and intracellular environments. Indeed, H 2 O 2 diffuses into bacterial cytoplasm and potentially causes damage of a variety of molecules [78]. Intracellular H 2 O 2 is also capable of triggering downstream signal pathways inducing pro-inflammatory cytokines production [79]. Generally, the treated and untreated macrophages are subjected to 100 μl of phenol red buffer containing peroxidase (5 mM glucose, 0⋅56 mM phenol red solution and 8⋅5 UI/ml HRP). Following incubation for 1 h at 37 • C, the reaction is stopped by addition of 10 μl of 1 M NaOH.
Absorbance is then determined at 620 nm using a microplate reader. The concentration of H 2 O 2 is determined based on the standard curve for H 2 O 2 [80].

Acid phosphatase activity/cellular lysosomal enzyme activity
During phagocytosis, macrophage cells acquire acid phosphatase by fusion with lysosomes. Thus, acid phosphatase functions as a signal enzyme for macrophage activation. To investigate the activity, treated cell supernatant is thoroughly removed and 25 μl of Triton X-100 (1%) along with 150 μl of p-nitrophenyl phosphate solution (1 mg/ml) are added. The mixtures are incubated for 1 h and 50 μl of NaOH solution (3 M) is added to terminate the reaction. Finally, absorbance is detected at 405 nm. The index of acid phosphatase activity is obtained according to the following equation [56]: Index of acid phosphatase activity = Absorbance of treatment/Absorbance of blank

Recognition of membrane receptors
To eradicate pathogens, macrophages must be able to distinguish between self-particles and infectious materials. The molecules mainly responsible for making this pivotal distinction are those of family of transmembrane glycol-protein receptors called TLRs. These receptors were named after the fruit-fly receptor Toll, which was recognized as contributing to innate immunity in adult flies [81]. To date, total 13 mammalian TLRs have been recognized, of which 12 functional TLRs (TLR1-TLR9, TLR11-TLR13) have been identified to be expressed in mice and 10 (TLR1-TLR10) in humans where TLRs 1-9 are conserved in both. Typically TLRs 1, 2, 4-6 and 10 are displayed in cell surface and the rest are localized to intracellular compartments such as ER, endosome, lysosome, or endolysosome. Cell surface TLRs recognize mainly microbial membrane components including lipoproteins, lipids and proteins [82]. Activation of TLR-4 results in triggering two dissimilar cascades such as myeloid differentiating primary response gene 88 (MyD88) dependent and MyD88 independent pathway. Stimulation of MyD88 led to generation of pro-inflammatory cytokines via triggering of a TF, NF-κB, (Fig. 2). Stimulation MyD88-independent pathway, on the other hand, led to synthesis of type 1 interferons (IFN)s [83]. Apart from TLRs, macrophages also contain mannose receptor (MR) that binds to microbial structure bearing mannose, N-acetylglucosamine and fucose on surface triggering endocytosis and phagocytosis [84]. Dectin-1 is another PRR that is expressed on macrophage surface and recognizes β-glucans in cell walls of fungi. Activation of the receptor results in phagocytosis, ROS production, synthesis of inflammatory cytokines and chemokines that in turn influences development of adaptive immunity [85]. Several other receptors such as scavenger receptor (SR), glucocorticoid receptor (GR) and complement receptor 3 (CR3) also play an important role in inflammation, innate immunity and host defence. To detect the involved receptor, polymerase chain reaction (PCR), western blotting and RNA interference (RNAi) techniques are the most prevalent methods.

RNA isolation, cDNA preparation, reverse transcriptase-PCR
After treatment, macrophage cells are lysed by 1 ml TRIzol reagent.
Then 200 μl chloroform is added, lysates are shaken vigorously by hand for 15 s and incubated at RT for 2-3 min. After centrifugation for 15 min, the aqueous phase is transferred to a fresh tube. RNA is washed thoroughly with isopropyl alcohol and 75% ethanol. The pellet is briefly dried and dissolved in RNase-free water. Total RNA yield is measured by 260 nm absorbance and its quality can be assessed by agarose gel electrophoresis [86]. Reverse transcription of RNA is carried out using reverse transcriptase, oligo (dT) 16 primer, dNTP and RNase inhibitor. The generated cDNA is amplified by PCR using primer set for specific gene ( Table 2 and Table 3) where β-actin or GAPDH are used as housekeeping genes. The cDNA is generally amplified by 35 cycles of denaturing at 94 • C for 45 s, annealing for 45 s, extension at 72 • C for 1 min and finally extended at 72 • C for 5 min. The products can be separated in 1.5% (w/v) agarose gels and visualized with UV light [393]. For each gel, ImageJ software (http://rsbweb.nih.gov/ij/index. html) might be applied for quantitative estimation of band intensity.     55 [114] (continued on next page)

Quantitative PCR (qPCR)
Quantitative PCR relies on real-time detection of amplified cDNA targets generated by successive rounds of PCR. Amplicons are detected based on fluorescence intensity which increases uniformly with the PCR product. However, the required equipment and reagents are more expensive than that of conventional PCR technology. To perform the assay, cDNA is prepared from each experimental set as described before and then amplified by using incorporation of SYBR green. Relative expression of specific genes can be calculated using 2− ΔΔ CT method [9,17,18,40,74,99,101,105].

Receptor detection by inhibitor treatment
Cells are adjusted to a concentration of 1 × 10 6 cells/ml in the exponential phase, loaded into 96-well plate (100 μl/well) and incubated for 24 h. Cells are pre-treated with TLR4-IN-C34, a drug that inhibits TLR-4, for 30 min, followed by incubation with sample. After the incubation period, cell supernatant is analysed for NO content by using Griess reagent [17]. Otherwise, the cells can be pre-treated with antibodies (5 μg/ml) of membrane receptors (SR, MR, GR, CR3, TLR-2, TLR-4 and Dectin-1) for 1-2 h to blockade the matched receptors. Then, above cells are cultured with sample at 37 • C in humidified chamber. The cells treated with sample in absence of antibody and the cells without any treatment are used as control groups. Then the activities of treated macrophages can be determined in terms of NO production in culture supernatant [9,63,99].

Western blot analysis
One of the disadvantages of transcriptome analysis is that presence of mRNA does not always precisely reflect protein level as many proteins are modified at post-translational level. The limitation can be overcome by performing western blotting method. For that, macrophage cells are grown in 6-well plate (1 × 10 6 cells/well) with stimuli or other treatment. Cells are lysed using cell lysis buffer (100-300 μl/well) prepared by mixing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4), 2 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,   Subsequently, the membrane is washed five times and incubated with an appropriate secondary antibody (HRP-conjugated goat anti-mouse or anti-rabbit IgG) (1:500) in 2% (v/v) PBS for 1-2 h. After washing the membrane with TBST three times for 10 min, the blots can be developed with chemiluminescence [95,123].

RNAi
RNA interference involves double-stranded RNA-mediated degradation of target mRNA. Such gene-specific suppression can be achieved by delivering of 21-23 nucleotide long interfering RNAs (siRNAs) [124]. To determine whether the mechanism of action of the investigating drug is mediated through TLR-4, MyD88 or TRIF, the mRNAs can be silenced using oligonucleotide sequences as described by Qi and Shelhamer [123]. The siRNA duplexes are transfected into macrophage cells which are then seeded at 0.2 × 10 6 cell/well in 24-well plate. After 24 h of transfection, the medium is changed with 1 ml of fresh DMEM and treated with stimuli. For RNA and protein extraction, the cells are harvested in TRIzol after 6 h and 12 h of treatment respectively [88]. The interference of TLR4, MyD88, and TRIF protein expression can be confirmed by immunoblotting using specific antibodies [123].

Assays on NF-κB protein
Nuclear factor kappa-B is a key pro-inflammatory TF that regulates transcription of numerous regulatory receptors, cytokines, chemokines and enzymes. The protein is activated when macrophages sense pathogens through multiple classes of PRR pathways [125]. The protein consists of homo-and hetero-dimers of five subunits, p65/RelA, RelB, c-Rel, p50/NF-κB1 and p52/NF-κB2. In resting cells, NF-κB localizes in cytoplasm via association with cytosolic IκB (inhibitor of NF-κB) proteins such as IκB-α, -β and -ε. Upon stimulation, IκB kinase (IKK) phosphorylates IκB which is then degraded by ubiquitin-proteasome system [126]. Subsequently, NF-κB dimer then translocates to nucleus, binds to its consensus sequence on promoter and enhancer of target genes and activates transcription of pro-inflammatory genes (Fig. 2). Monitoring NF-κB movement hence is a widespread method to measure activity of the TF. Two techniques have been developed to test the translocation: 1) Cell fractionation isolating cytoplasmic and nuclear fractions where NF-κB is quantified using anti-phospho-NF-κB p65 by western blotting [95]. 2) Image-based tracking of NF-κB where dynamics of the protein are monitored either by antibody staining or using a fluorescent protein fused to the TF [126,127].

Immunofluorescence study to visualize NF-κB nuclear localization
Immunofluorescence assay (IFA) is a microscopic technique to detect and visualize any protein expressed in cells via antigen-antibody reaction. To perform the assay, treated macrophage cells, grown on glass cover slips, are fixed with 4% paraformaldehyde in PBS for 15-20 min at RT. Cells are then repeatedly washed with PBS (5 min each) and permeabilized with 0.3-2% Triton X-100 in PBS for 5 min. Cells are again washed and then blocked with 1-5% BSA in PBS for 1 h. Cells are then incubated with primary antibodies against NF-κB p65 (1:200 or 1:400) overnight at 4 • C. After washing three times, cells are incubated with appropriate secondary antibodies (1:500) of fluorescent dye-conjugated anti-rabbit IgG or fluorescent dye conjugated goat anti-rabbit IgG plus 10 μg/ml of Hoechst 33342 for 1-2 h at RT in dark. Cells are then washed three times with PBS and DAPI (1 μg/ml in PBS) is used to stain nuclei. Finally, sample on cover slips may be mounted with nonhardening, aqueous-based compound, available commercially, to provide a semi-permanent seal for storage of slide preparations. The observations can be performed on a fluorescence microscope with 100 × magnification capturing micrographs [128].

Inhibition of NF-κB using specific inhibitors
BAY 11-7082 (BAY) is an inhibitor of NF-κB and thus used to verify action of the TF mediated signalling pathway. Research has established that BAY strongly suppresses production of NO, prostaglandin E2 (PGE2), and tumor necrosis factor-α (TNF-α). It also reduces the translocation of p65 and its upstream signalling events such as

Transient transfection and luciferase reporter assay
Macrophage cells are cultured in a 60-mm dish. After confluence, they are transfected with pNF-κB-Luc plasmid comprising NF-κB responsive region followed by receptor gene firefly luciferase along with certain antibiotic resistant gene. After 48 h, cells are cultured in medium supplemented with the antibiotic for at least one week. Clone revealing the strongest luciferase activity due to LPS treatment is seeded (4 × 10 5 cells/well) in 24-well plate for overnight. After treatment with drug, cells are harvested and extracted with lysis buffer (100 μl). Twenty μl of the lysate is then mixed with 100 μl luciferin and the luminescence is measured. Results can be described as relative luciferase activity (fold difference in comparison with negative control) [75,92,114,128].

Electrophoretic mobility shift assay (EMSA)
Electrophoretic mobility shift assay is classically used to detect DNA binding proteins. Principle of the technique is that protein-DNA complex moves more slowly through a polyacrylamide gel than the corresponding free linear unbound DNA. This high sensitivity and simple to perform method can be used for studying DNA and protein interactions [130]. The assay traditionally requires radioactive probes; however, biotin or fluorescent probes are now available. To perform the assay, treated and untreated macrophage cells are centrifuged at 800×g for 5-10 min at 4 • C. Cells are lysed with 300 μl pre-cold hypotonic buffer (5 μl phosphatase inhibitor, 10 μl phenylmethylsulfonyl fluoride (PMSF) and 1 μl dithiothreitol or DTT in 1 ml of buffer). The tube is flicked with finger, bathed on ice for 10 min and shaken for 10 s. The suspension is centrifuged at 800×g for 5 min and the supernatant is discarded immediately. The buffer (400 μl) is added to wash the pellet and the tubes are again centrifuged at 2,500×g for 5 min. The supernatant is discarded and the precipitate is saved. Lysis buffer (200 μl) (10 μl PMSF, 5 μl phosphatase inhibitor and 1 μl DTT in 1 ml buffer) is added to the precipitate, bathed on ice for 20 min and centrifuged at 20,000×g for 10 min or 14,500×g for 15 min. Precipitate is discarded and the supernatant is stored at − 80 • C as nuclear protein extract. The protein concentration can be measured using BSA. NF-κB or p65 (5 ′ AGTTGAGGGGACTTTCCCAGGC3 ′ ) probe is labelled with biotin or [ γ -32P]-ATP. NF-κB probe (0.5 μl) and nuclear protein extract (40 μg protein) are incubated in binding reaction for 20 min at RT. Non-denaturing 6.5% PAGE is carried out and electrotransfer is carried out in TBE buffer. Gel is dried and subjected to autoradiography or chemiluminescence [23,95].

RelA translocation assay
In order to interpret NF-κB activation, a nuclear translocation assay can be performed. The method is based on quantification of the amount of translocated p65 subunit or RelA in nucleus and comparison with that of remaining in the cytoplasm. Accordingly, treated and untreated macrophages are fractionated into nuclear and cytoplasmic fractions, supernatants are collected separately and p65 is quantified in each by Western blot technique [131].

Determination of cytokines and chemokines expression
Cytokines are a diverse group of low molecular weight protein that act as pro-or anti-inflammatory factors at pico or nanomolar levels to regulate inflammation and cellular activities. Whilst, inflammatory chemokines are generated in response to an inflammatory stimulus and facilitate an immune response. Binding of a cytokine or chemokine ligand triggers a cascade of events regulating various cellular functions such as phagocytosis, cytokine secretion, cell activation and cell proliferation, among others. These potent signalling molecules thus are a cornerstone of any investigation that deals with inflammation [132]. Following macrophage activation, NF-κB promotes gene expression of a wide variety of cytokines such as interleukin (IL)-1β, IL-2, IL-6, IL-12, TNF-α and granulocyte-macrophage colony-stimulating factor (GM-CSF); chemokines such as IL-8, macrophage inflammatory proteins (MIP)-1, monocyte chemoattractant protein-1 (MCP-1) or C-C motif chemokine ligand (CCL)2, regulated on activation normal T cell expressed and secreted (RANTES) or CCL5 and eotaxin; and inducible effector enzymes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) (Fig. 2) [133]. NF-κB activates its own inhibitor, IκB-α, as well which then translocates to the nucleus, binds to promoter bound NF-κB, and returns it to cytoplasm in an inactivate state. Such post-induction repression process eventually terminates transcription [134]. Overall, NF-κB serves as a 'rapid acting' primary TF which can regulate host's early innate immune response to infection and also associated with chronic inflammatory states and multiorgan failure [133]. Several methods have been developed to measure cytokine levels; amongst them ELISA is routinely used.

Quantitative analysis by ELISA
The procedure exploits specificity of antibodies and uses them to quantify an analyte of interest with remarkable sensitivity. To perform the method, macrophage cells are seeded into 6 or 12-well plates (1 × 10 6 cells/well). After treatment, culture media are transferred into 96well plates coated with 100 μl of antibody solutions of cytokines at 37 • C for 1 h. Following four washes, 100 μl of HRP solution is added and the plates are left to stand for 40 min. The solution is washed repeatedly and 3,3 ′ ,5,5 ′ -tetramethylbenzidine (TMB) substrate solution (100 μl) is added. The reaction mixtures are incubated in the dark and a stop solution is added after 15 min. After 5 min, the absorbance is read at 450 nm on a microplate reader. The cytokine levels are computed according to standard curves and generally represented in picograms per milliliter [127].

Evaluation of expression of co-stimulatory molecules
Macrophage cells play an important role as an interface between innate and adaptive immunity. Following activation, the cells express markers (CD68, CD86, CD80, F4/80, CD11b, CD40, B7-1 and B7-2) known as co-stimulatory molecules that are responsible for functions such as antigen processing and presentation to antigen-specific T cells. CD40 interacts with CD40L, which is known to play key roles in activation and differentiation of B cells. Thus, increase in expression of these surface molecules indicates enhanced immune response [95,138].
To perform the assay, cells are collected following treatment and then washed with PBS. The cell surfaces are blocked with 5% goat serum for 15 min or staining buffer comprising 10% normal mouse serum for 20 min on ice and washed twice. Subsequently, the cells are incubated with monoclonal antibodies against CD80, CD40, CD86 and corresponding fluorescent markers for around 30 min at 4 • C. Following washing, the cells are fixed by 1% paraformaldehyde in PBS. The fixed cells are subjected to flow cytometry and minimum 5000 cells are analysed for each sample [95].

cDNA microarray
cDNA microarray technology leads to identification of specific genes and allows researchers to compare profiles of gene expression in normal versus pathological conditions [139]. In brief, macrophage cells are placed at a density of 5 × 10 5 cells/ml in medium and treated with stimuli. The total RNA is extracted from each treatment and utilized for cDNA synthesis, labelling and microarray hybridization. The resulting localized concentrations of fluorescent molecules are noticed and quantitated [54].

Conclusion and future prospects
In recent years, immunotherapy has become of great interest to researchers, clinicians and pharmaceutical companies as it has the high potential to revolutionize treatment regimens. In that note, in vitro assays have shown their usefulness as an initial platform for drug development elucidating mode of action. However, techniques related to transcriptome analysis, estimation of protein expression and detection of post-translational modification are comparatively expensive, hindering many researchers to perform the assays. Thus, attempt should be taken to standardize more cost-effective and easier to perform procedures to make way wider and smoother for testing and discovering novel therapeutic agents.

CRediT authorship contribution statement
Somanjana Khatua: performed the literature search, wrote the manuscript. Jesus Simal-Gandara: conceived the idea and revised the manuscript. Krishnendu Acharya: conceived the idea and revised the manuscript.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.