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The kinetics of promonocytes and monocytes in the bone marrow.

The mononuclear phagocytes of the bone marrow can be classified into two cell types, promonocytes and monocytes. The present study was performed to establish whether the promonocytes are the progenitors of the monocytes and to determine the kinetic characteristics of the promonocytes and monocytes in the bone marrow compartment. Both in vitro labeling studies with thymidine-3H and determination of the relative amount of DNA in the nuclei of individual cells showed that under normal steady-state conditions the promonocytes are proliferating cells and the monocytes, nondividing cells. In vivo labeling studies provided further evidence that the promonocytes are the progenitor cells of the monocytes. During the first 24 hr after labeling, the promonocytes showed a constant high level of labeling (about 70%). The mean grain count of these cells decreased with time. The labeling index of the monocytes of the bone marrow increased during the first 24 hr after in vivo labeling, but during the same period the mean grain counts remained almost constant, with values amounting to about half those of the promonocytes during the first 6 hr of the experiment. The data concerning the labeling indices and the percentage distribution ratio of the promonocytes and monocytes in the bone marrow, and the labeling indices of the peripheral blood monocytes are used to construct a model population. The results lead to the conclusions that the promonocytes are multiplicative cells and that both daughter cells arising from the division of a promonocyte are monocytes. The DNA-synthesis time found for the promonocytes is 13.6 hr. From this value, the average generation time was computed to be 19.5 hr.

Peripheral Blood Monocytes.--Monocytes of the peripheral blood were studied in smears prepared from tail-vein blood. The slides were fixed in absolute methyl alcohol for 30 rain.
Labeling with Thymidine-3tt.--The in vitro labeling of the mononuclear phagocytes of the bone marrow was performed by incubating the cells for a given period in the culture medium, which contained 0.1 #Ci/ml methyl thymidine3H (specific activity 6.7 mCi/mnmle, New England Nuclear Corp., Boston, Mass.). The culture medium used to flush the femur shafts already contained thymidine-3H. After the initial incubation period of 2 hr, the cover slip preparations were washed and again cultured. The cells on the cover slips were incubated in the presence of thymidine-3H for a total period of 2, 6, 24, or 48 hr, after which the cover slips were washed five times with medium 199 to ensure the removal of all thymidine-3H. According to the requirements of the experiment, the slides were then reineubated in the nonradioactive culture medium or quickly air dried, fixed with absolute methyl alcohol, and prepared for radioautography.
In vivo labeling was done by the administration of one intravenous injection of thymidine-~H, in a dose of 1/~Ci/g body weight. The bone marrow was harvested from 2-96 hr after the injection. For each animal, one of the duplicate bone marrow cultures was incubated for 6 hr and the other for 48 hr, as described elsewhere (1). After incubation, the cover slips were washed, rapidly air dried, fixed in absolute methyl alcohol, and prepared for radioautography.
Serial injections of thymidine-nH were also applied to a group of mice to determine the DNA-synthesis time of the promonocytes. These mice received 1, 2, or 3 intravenous injections of thymidine-SH in a dose of 1 #Ci/g body weight, with a 2 hr interval, at the time points 0, 2, and 4 hr. Bone marrow was taken 1 and 2 hr after each injection and cultured for 6 hr. The labeling index for each time point represents the mean of values for three mice.
Radioautography.--Radioautography was performed with the Ilford Nuclear Research Emulsion K5, in gel form (Ilford Ltd., Ilford, Essex, England) (1). The developed slides were stained with Giemsa stain (1). Bone marrow cultures were stained for about 20 rain and blood smears for about 90 rain.
All cells with three or more silver grains over the nucleus were considered as labeled, since background levels on preparations from nonlabeled animals showed less than three grains per nucleus.
At each time point, at least 200 mononuclear phagocytes were counted in duplicate bone marrow cultures deriving from one mouse. The labeling index for each time point in the graphs or tables represents the mean value of at least two mice but in many cases of four to eight mice.
The total percentage of labeled cells (Table I and Fig. 4) was calculated by using the labeling indices and the percentage distribution of promonocytes and monocytes on the same slide.
The mean grain count at each time point was calculated from the grain counts determined in 100 labeled cells.
Determination of the DNA Content of the lPromonocytes and Monocytes.--Bone marrow cell suspensions were cultured for 6 hr, washed, air dried, and fixed with absolute methyl alcohol. After the cells on the cover slip had been stained by the Feulgen reaction (3), the DNA content of the nuclei was determined ultramicrospectrophotometrically (Zeiss Ultramicrospectrophometer I, Carl Zeiss, Loberkochen, W. Germany [4]). As a reference for the diploid value, the DNA content of the polymorphonuclear cells on the same slide was used.
Identification oJ the Cells.--The criteria used for the morphological differentiation of the mononuclear phagocytes of the bone marrow into promonocytes and monocytes has been discussed in the preceding paper (2). Promonocytes have a diameter of 14-20 tz, a basophilic cytoplasm, and a relatively large indented or folded nucleus; monocytes have a diameter of 11-14 #, a pale-blue cytoplasm, and an indented horseshoe-shaped or reniform nucleus. The ratio between promonocytes and monocytes is about 1:5 (2).

In Vitro Labeling of Promonocytes and Monocytes of the Bone Marrow.-
After incubation of bone marrow cells for 2 hr in a culture medium containing thymidine-~H, and reincubation in a nonradioactive medium for 4 hr, 47.6 % (range 38.5-66.6%) of the promo:aocytes were labeled, but no labeled monocytes were present (Table I). Fig. 1 shows a labeled promonocyte which was allowed to phagocytize Staphylococcus albus (2). When the bone marrow cells were incubated during the entire 6 hr culture period in the presence of thymidine-all, the monocytes were again unlabeled, and 54.0% (range 39.0-63.0%) labeled promonocytes were found; in the 24-hr cultures 50.3% (range 40.5-58.0%) of the promonocytes were labeled ( Table I). Labeling of a small per-Fro. 1. A promonocyte labeled in vitro and a doughnut cell. The promonocyte has phagocytized Staphylococcus albus. X 1200.
centage of monocytes (range 0-6.9 %) occurred only in bone marrow cultures incubated for 24 hr (Table I).
After a culture period of 48 hr, the classification into promonocytes and monocytes could not be made because all the cells had the morphology of macrophages (2). With incubation during the total culture period of 48 hr in a medium containing thymidine-3H, 28.2% (range 19.5-41.0%) of the mononuclear phagocytes were labeled (Table I). When the bone marrow cells initially incubated in the radioactive medium for only 2 or 6 hr were cultured for 48 hr, 22.5% (range 18.5-26.5%) and 20.3% (range 19.5-21.5%) of the mononuclear phagocytes were labeled respectively.
To compare the percentages of labeled mononuclear phagocytes found after 48 hr of incubation with those of the 6-and 24-hr cultures, the total percentage of labeled cells from these cultures was calculated, taking into account the percentage distribution of the promonocytes and monocytes on the same slide. These mean values ranged from 10.8 to 15.4 %, which is lower than the labeling indices of the 48-hr cultures ( Table I).
The mean grain count of the promonocytes labeled in vitro for 2 hr was 44.3 grains per cell after a total incubation time of 6 hr. This value decreased to 31.4 grains per cell after an incubation period of 48 hr (Table II). Since no th)anidine-'~H was present after the first 2 hr of incubation, the labeled cells found at 48 hr may be considered to derive from the initially labeled promonocytes. Sinfilar results were obtained when the bone marrow cells were incubated for 6 hr in the presence of thymidine-~H. The mean grain counts of the pro- monocytes were found to be higher, but this also dropped with longer incubation periods in a nonradioactive medium (Table II). The results of these in vitro labeling studies show that the promonocytes incorporate thymidine-3H, which indicates that these are dividing cells. However, the monocytes of the bone marrow did not synthesize DNA. This is in accordance with earlier results, which demonstrated that the monocytes of the peripheral blood were not labeled after incubation for 24 hr in the presence of th)anidine-3H (1). If the absence of in vitro labeling of the monocytes reliably reflects the state of DNA synthesis of these cells in vivo, then monocytes must be nonproliferating cells. The small percentage of labeled monocytes found in the 24-hr cultures probably originated from promonocytes dividing in vitro, as discussed below.
The labeling index of the promonocytes appears to be independent of the time of incubation with thymidine-3H. However, after an incubation period of 48 hr the total percentage of labeled cells increased 1.5-1.8 times as compared to the calculated values for the 6-and 24-hr cultures (Table I). This significant increase (P < 0.005) could be due to the death of unlabeled cells or to division of the labeled promonocytes. The decrease of the mean grain counts of the labeled cells with time of incubation indicates that at least some of the promonocytes divide during incubation in vitro.  cells with thymidine-3H. I n contrast, 80% of the promonocytes had a D N A content that was more than diploid and less than or equal to the tetraploid value. These D N A values agree with the in vitro and in vivo labeling data, which showed that 50--70% of the promonocytes incorporate thymidine-3H.

In Vi~o Labeling of Promonocytes and Monocytes of the Bone Marrow and Peripheral Blood Monocytes after one Injection of Thymidine-3tt.--The in vivo
labeling studies demonstrated that 2 hr after a single injection of thymidine-3H, 68.7 % of the promonocytes were labeled (Fig. 2). This labeling index remained almost constant during the first 24 hr; thereafter, the percentage of labeled promonocytes decreased slightly to a value of 52.6% at 72 hr, and at 96 hr the labeling index had dropped to 29.3% (Fig. 2). Since promonocytes are dividing cells, this decrease of the labeling index at 96 hr can probably be ascribed to the fact that at later time points some proportion of the promonocytes, all of which derive from initially labeled cells, had grain counts below the 3 grain threshold and thus were not considered positive. Fig. 3 shows a promonocyte and a monocyte labeled in vivo.
The labeling index of the monocytes in the bone marrow 2 hr after the thymidine-3H injection was only 1%, and reached its highest value, i.e. 33.3 % labeled monocytes, at 24 hr. Thereafter, this percentage decreased gradually to 12.3 % at 96 hr (Fig. 2).
The labeling indices of the peripheral blood rnonocytes, determined in the same mice, are also presented in Fig. 2. This curve demonstrates that 2 hr after a single injection 2 % of the monocytes were already labeled, and that the highest value (59 %) was attained at 48 hr. These results agree with the findings in a previous study (1).
The mean grain counts of the labeled promonocytes and bone marrow monocytes (Table III) gave for the promonocytes a value of 21.6 grains per cell 2 hr after the thymidine-~H injection. This value decreased with time, and at 72 hr amounted to 5.9 grains per cell. At 6 hr the mean grain count of the monocytes was 13.9 grains per cell, i.e. half the initial mean grain count of the promonocytes, which may indicate that monocytes are derived from promonocytes after division.
At each time-point, a second bone marrow culture of the same mouse was also incubated for 48 hr. After this period of incubation, the mononuclear phagocytes of the bone marrow can no longer be classified as promonocytes and monocytes because morphologically all the cells are macrophages (1,2). In these cultures, 15% of the mononuclear phagocytes were labeled 2 hr after thymidine-3H injection. The labeling index increased with time until the highest value, i.e. 47 % labeled cells, was reached at 24 hr. Thereafter, the labeling index decreased to 15 % at 96 hr (Fig. 4). Similar values were found in earlier experiments (1). From both the data on the labeled promonocytes and monocytes in the 6-hr cultures and the ratio of promonocytes to monocytes for each slide, the total percentage of labeled cells was calculated for each time point. These percentages are shown in Fig. 4. Since these two curves coincide almost completely, there is excellent agreement between the percentages of labeled cells found in the 6-and 48-hr cultures. These results therefore provide additional support for the validity of the classification of the mononuclear phagocytes of the bone marrow into promonocytes and monocytes.
Both the total number of labeled monocytes formed after a single injection of thymidine-3H and their distribution over the bone marrow and blood compartments can be calculated, since the size of these two pools can be determined (see Discussion: the monocyte pool in the peripheral blood amounts to 1.1 X l0 G cells and the bone marrow contains 2.4 X 106 monocytes). At each time point, the number of labeled bone marrow monocytes will be the product of the labeling index and the number of monocytes in the bone marrow compartment (Ibm X Xbm). The number of labeled monocytes released into the pc-ripheral blood is at each time point the product of the labeling index and the total number of peripheral blood monocytes plus the number of monocytes that have already left the peripheral blood compartment (Ibl)< Nul + 0.6932/ T 1/6 of t Ibl X Nbl dt) (Fig. 5). The total number of monocytes produced is the sum of the labeled monocytes in the bone marrow and the monocytes released nto the peripheral blood. Accordingly, during the first 18 hr after labeling the  [1].) The total number of monocytes produced is the sum of the monocytes in the bone marrow compartment and the monocytes released into the peripheral blood.
number of labeled monocytes in the bone marrow compartment increases by 4.1 X 104 cells/hr and 2.0 X 104 monocytes/hour are released into the peripheral blood. However, in the period from 18-24 hr 5.2 X 104 labeled monocytes/hr are released into the peripheral blood and the increase of labeled bone marrow monocytes is only 1.6 X 104 cells/hr. After 24 hr the formation of labeled monocytes levels off (Fig. 5).
Serial Labeling with Thymidine-3tt for the Determination of the DNA-Synthesis Time and the Generation Time of Promonocytes.--Mice were labeled one, two, or three times by intravenous injection of thymidine-~H at 2-hr intervals.
In the bone marrow samples harvested 1 and 2 hr after each injection, the labeling index of the promonocytes and monocytes was determined. For the promonocytes harvested 1 hr after the first injection of thymidine-3H, this value was 71.3% (Table IV); 2 hr after the third injection, the index had increased to 92.8 %. The initially very low (0.3 %) percentage of labeled monocytes had increased to 11% 2 hr after the third injection.
The percentage of promonocytes entering DNA synthesis during the 2-hr periods between the injections can be determined from the increase of the labeling indices. For the calculation of the DNA synthesis time, the mean of the labeling indices of the promonocytes found 1 and 2 hr after the first injection of thymidine-3H (69.8 %) and the labeling index found 1 hr after the second injection (80.1%) were used. The increment of the labeling index during this 2 hr interval between the thymidine-3H injections amounted to 10.3% (i.e. 5.15 %/hr).
The DNA-synthesis time can be computed by dividing the initial labeling index by the increment of the labeling index per hour (5). The average I)NAsynthesis time of the promonocytes is then 69.8/5.15 --13.6 hr. This approach is valid if the following criteria are satisfied: studies must be done under normal steady-state conditions, the interval between the thymidine-~I-I injections must be shorter than the DNA-synthesis time, and the whole generation of dividing cells, i.e., the promonocytes, must be recognizable as one cytological class (4).
The generation time, i.e. the time between two consecutive divisions, can be computed by dividing the DNA-synthesis time by the initial labeling index (5). This gives an average generation time for the promonocytes of (13.6 X 100)/69.8 = 19.5 hr. DISCUSSION Previous investigations indicated that the monocytes of the peripheral blood derive from a precursor cell in the bone marrow (1). In the preceding paper it was shown that in 6-hr cultures the mononuclear phagocytes of the bone marrow can be classified morphologically into two cell types, promonocytes and monocytes (2). The present study was performed to investigate the validity of the hypothesis that the promonocytes are the progenitors of the monocytes and to determine the kinetic characteristics of the promonocytes and the monocytes in the bone marrow compartment.

Proliferation Pattern of the Promonocytes.--The results of the in vitro labeling
studies permit the conclusion that the promonocytes are proliferating cells, since about 50 % of these cells are labeled after incubation with thymidine-SH. However, under normal steady-state conditions the monocytes of the bone marrow appear to be nondividing cells, as was previously shown to be true for blood monocytes (1). These conclusions are supported by the observation that the nuclei of the monocytes have a diploid amount of DNA, whereas the majority of the promonocytes have a DNA content that is more than diploid and less than or equal to tetraploid.
Further evidence supporting this conclusion has been provided by the in vivo labeling studies. During the first 24 hr after a single injection of thymidine-3H, the promonocytes showed an almost constant and high labeling index. The labeling index of the monocytes of the bone marrow increased during the first 24 hr after the administration of thymidine-*H, but the mean grain count remained almost constant during that period, being about half that of the promonocytes during the first 6 hr of the experiment. Since monocytes are nondividing cells, these findings indicate that the monocytes derive from the promonocytes after the latter have divided once. Now let us consider the question of whether promonocytes are multiplicative cells, i.e. cells only capable of a rather limited number of mitoses, or are selfreplicating cells, i.e., cells capable of continuous self-perpetuation (5). For the purposes of this discussion, we can take a model population of 1000 bone marrow mononuclear phagocytes; the total number of labeled promonocytes and bone marrow monocytes is calculated on the basis of the percentage distribution of the promonocytes and monocytes and their labeling indices at each time point. The total number of labeled peripheral blood monocytes corresponding to this model of the bone marrow population could also be calculated, since the relative size of the monocyte pool in the peripheral blood is known (see below). The results of these calculations (see Table V) show that the total number of labeled promonocytes remains constant during the first 24 hr after labeling, and that the total number of labeled bone marrow and peripheral blood monocytes increases with time.
2 hr after labeling, this model population will contain 110 labeled promonocytes. If the promonocytes were multiplicative cells, after the first division these 110 cells would give rise to 220 labeled monocytes having half the grain count shown by the promonocytes at 2 hr. Actually, these values were found 12 hr after labeling. At that time point the total number of labeled bone marrow plus peripheral blood monocytes (the latter being cells that have recently left the bone marrow) amounts to 215 cells. This would then indicate that the DNA-synthesis time, G2-phase, and mitotic time together must be roughly 12 hr; this is compatible with the average DNA-synthesis time of 13.6 hr computed from the data obtained after repeated labeling with thymidine-3H. (The minimum duration of the G~-phase and mitosis must be 2 hr or less, since labeled monocytes were observed in the bone marrow and peripheral blood as early as 2 hr after labeling.) After one more division, which requires the full generation time of 19.6 hr, the total number of labeled monocytes can be expected to have roughly doubled again, if monocytes having left the peripheral blood during that period are not taken into consideration. Actually, the total number of labeled bone marrow plus peripheral blood monocytes found at 24 and 48 hr lies just a little higher than the expected value. The labeled promonocytes observed at these time points must derive from cells of a preceding compartment, probably after division.
of that of the promonocytes at 2 hr. This is observed for the monocytes at 6 hr, but at that time point the mean grain count of the promonocytes is not halved and furthermore, the observed DNA-synthesis time of the promonocytes is more than 6 hr. For both possibilities, an estimate of the DNA-synthesis time can be made from the flux of labeled promonocytes into the monocyte pool of the bone marrow and from there into the peripheral blood. If we consider the period between 2 and 12 hr for the model population (Table V), there would be an influx of 198 labeled monocytes during that interval, or 20 labeled monocytes per hour. If the promonocytes are multiplicative cells, after division both daughter cells would be monocytes. An influnx of 20 labeled monocytes per hour would then represent a flux into and out of DNA synthesis amounting to 20/2 = 10 promonocytes per hr. Since the number of promonocytes in DNA synthesis amounts to 110 cells, the DNA synthesis time can be computed to be 110/10 --11.0 hr. If the promonocyte is a self-replicating cell, then numerically only one daughter cell of a divided promonocyte would become a monocyte and the other cell would remain a promonocyte. In that case, 20 promono-cytes per hour would flow in and out of DNA synthesis. The DNA-synthesis time would then be 110/20 = 5.5 hr.
The results of a separate experiment in which repeated labeling of the mice with thymidine-3H was applied, show that the average DNA synthesis time is 13.6 hr. This is compatible with the DNA-synthesis time of 11.0 hr estimated for the assumption that both daughter cells of the promonocyte are monocytes. If promonocytes were self-replicating cells the estimated DNA-synthesis time would have been half as long. It may therefore be concluded that the promonocytes are multiplicative ceils; the division of one promonocyte gives rise to two rnonocytes (Fig. 6). In all probability the promonocytes divide only once. The pool of promonocytes is maintained by the influx of cells from a pool of precursor ceils, whose identity is not yet known. These precursor cells probably do not stick to glass and would therefore escape detection with the methods currently used. The rather constant labeling index of the promonocytes during the first 24 hr after the administration of thymidine-3H suggests, however, that the labeling index of the precursor cell must be rather similar to that of the promonocytes.
Size of the Promonocyte and Monocyte Pools in the Bone Marrow.--We may now consider the size of the promonocyte and monocyte pools in the bone marrow and of the monocyte pool in the peripheral blood. Since mouse blood contains 350 monocytes per mm 3 (6) and the blood volume of mice weighing 25 g is about 3 ml (7), the total number of monocytes in the peripheral blood must amount to 10.5 X 105 cells. From this value, the total number of monocytes in the bone marrow can be calculated if all monocytes formed in the bone marrow are assumed to enter the peripheral blood. Since the average transit time of the monocytes in the peripheral blood is 32 hr (1), under normal steady-state conditions the entry of monocytes from the bone marrow into the peripheral blood compartment would be (10.5 X 105)/32 = 3.3 X 104 cells per hr. Promonocytes being multiplicative cells with a generation time of 19.5 hr, one promonocyte would give rise to 2 X 1/19.5 = 0.1 monocyte per hr. The production of 3.3 X 104 monocytes per hr would then require 3.3 X 105 promonocytes. Since the population of mononuclear phagocytes of the bone marrow consists of 15.9% promonocytes and 84.1% monocytes (2), the calculated total number of monocytes in the bone marrow would be 1.8 X 106 cells. Actually, we found that one femur contains 1.2 X 107 nucleated cells of which 1.2 % are monocytes. Since the bone marrow in two femurs accounts for 11.8 % of the total bone marrow mass (8), the total number of monocytes in the bone marrow of the mouse must be 2.4 X 106 cells, which agrees reasonably well with the calculated value. On the basis of the average of the calculated and observed values, the size of tbe monocyte pool of the bone marrow is about two times greater than the peripheral blood monocyte pool.

Kinetics of Promonocytes and Monocytes in the Bone Marrow Compartment.-
We may now turn to the transit of cells through the promonocyte and monocyte pools of the bone marrow. If we assume one division in the promonocyte pool, the influx of cells from the pool of precursor cells into the promonocyte pool would be half the efflux from the latter pool. Since the hourly production of monocytes is 3.3 X 104 cells, the influx into the promonocyte pool will amount to 1.7 X 104 cells per hr. The relative influx of newly formed cells into the bone marrow monocyte pool can be calculated from the rise of the labeling index of bone marrow monocytes between 2 and 24 hr after labeling, which amounts to 32 %, i.e., 1.5 % per hour. Since a model population of 1000 bone marrow mononuclear phagocytes contains about 850 monocytes, the entry of new monocytes will amount to 12.8 cells per hr. Roughly similar results are obtained when these calculations are applied to the 150 promonocytes of the model population. As the promonocytes are multiplicative cells with a generation time of 19.5 hr, 150/19.5 X 2 = 15.4 cells per hr will leave the promonocyte pool and enter the bone marrow monocyte pool; this agrees with the above calculation of the influx. Since the monocyte pool contains 850 cells, the calculated value of the turnover time of the monocytes in the bone marrow compartment is 850/15.4 = 55.2 hr. If the bone marrow monocytes were homogeneous and left the bone marrow according to a pipeline pattern, there would be a delay of 55.2 hr before labeled monocytes appeared in the peripheral blood. However, in the present and earlier experiments (1), labeled monocytes appeared in the peripheral blood during the first hours after labeling.
During the first 18 hr after labeling, per hour twice as many newly formed monocytes appear in the bone marrow compartment as in the peripheral blood, but in the next 6 hr the release of monocytes per hour into the peripheral blood is about 3 times greater than the increase of labeled monocytes in the bone marrow (see Fig. 5). This suggests that kinetically the bone marrow monocytes are not homogeneous. Conceivably, one fraction of the bone marrow monocytes is fed into the peripheral blood rapidly, whereas another fraction arrives there after some delay. SUMMARY The mononuclear phagocytes of the bone marrow can be classified into two cell types, promonocytes and monocytes. The present study was performed to establish whether the promonocytes are the progenitors of the monocytes and to determine the kinetic characteristics of the promonocytes and monocytes in the bone marrow compartment.
Both in vitro labeling studies with thymidine-3H and determination of the relative amount of DNA in the nuclei of individual cells showed that under normal steady-state conditions the promonocytes are proliferating cells and the monocytes, nondividing cells.
In vivo labeling studies provided further evidence that the promonocytes are the progenitor cells of the monocytes. During the first 24 hr after labeling, the promonocytes showed a constant high level of labeling (about 70%). The mean grain count of these cells decreased with time.
The labeling index of the monocytes of the bone marrow increased during the first 24 hr after in vivo labeling, but during the same period the mean grain counts remained almost constant, with values amounting to about half those of the promonocytes during the first 6 hr of the experiment.
The data concerning the labeling indices and the percentage distribution ratio of the promonocytes and monocytes in the bone marrow, and the labeling indices of the peripheral blood monocytes are used to construct a model population. The results lead to the conclusions that the promonocytes are multiplicative cells and that both daughter cells arising from the division of a promonocyte are monocytes.
The DNA-synthesis time found for the promonocytes is 13.6 hr. From this value, the average generation time was computed to be 19.5 hr.
The helpful advice of Dr. S. A. Killmann (Rigshospitalet, Copenhagen) and the valuable discussions with him are gratefully acknowledged. The microspectrophotometry was kindly performed by Dr. P. Ernst, Rigshospitalet, Copenhagen.