Double labeling with iodo- and bromodeoxyuridine for cell kinetics studies

The rate of progression through the cell cycle was determined in five human glioma cell lines by a new sequential immunohistochemical staining technique. The cells were labeled first with iododeoxyuridine (IdUrd) for 1-3 hr and then with bromodeoxyuridine (BrdUrd) for 30 min. Labeled cells were identified with Br-3, a monoclonal antibody that recognizes only BrdUrd, and with IU-4, an antibody that recognizes both IdUrd and BrdUrd. Each slide was stained sequentially, first with the immunoperoxidase method for Br-3 and then with the alkaline phosphatase-anti-alkaline phosphatase method for IU-4. Cells that were positive only for IU-4 represented the fraction of S-phase cells that passed into the G2 phase during the period of incubation with IdUrd. The rates of progression measured by this method were constant in each cell line and resulted in smaller standard errors than were obtained by measurements from specimens stained singly for IdUrd and BrdUrd in different slides. The duration of the S-phase calculated from this fraction in the five cell lines ranged from 8-13 hr; the estimated potential doubling times were 25-32 hr and were very similar to the actual doubling times.     

Flow cytometric estimation of cell cycle parameters using a monoclonal antibody to bromodeoxyuridine

An estimation of cell kinetic parameters was made by simultaneous flow cytometric measurements of DNA and bromodeoxyuridine (BrdUrd) contents of cells. The procedure described in this paper involves the incorporation of BrdUrd by S phase cells, labeling the BrdUrd with an indirect immunofluorescent technique using a monoclonal anti-BrdUrd antibody, and staining DNA with propidium iodide (PI). The amount of incorporated BrdUrd in HeLa cells was proportional to that of synthesized DNA through S phase. For all cell lines examined, the pattern of BrdUrd incorporation was essentially the same and the rate of DNA synthesis during S phase was not constant. The bivariate BrdUrd/DNA distributions showed a horse-shoe pattern, maximum in the mid S phase and minimum in the early and late S phases. Furthermore, the durations of cell cycle (Tc) and S phase (Ts) were estimated from a FLSm (fraction of labeled cells in mid S phase) curve that was generated by plotting the percentage of BrdUrd pulse-labeled cells in a narrow window defined in the mid S phase of the DNA histogram. The values of these parameters in NIH 3T3, HeLa S3, and HL-60 cells were in good accordance with the reported data. This FCM method using the monoclonal anti-BrdUrd antibody allows rapid determination of both cell cycle compartments and also Ts and Tc without the use of radioactive DNA precursors.     

Alteration of the Cloudman melanoma cell cycle by prostaglandins E1 and E2 determined by using a 5-bromo-2'-deoxyuridine method of DNA analysis

Prostaglandins (PGs) E1 and E2 stimulate tyrosinase activity and suppress the proliferation of Cloudman S91 melanoma cells by altering their progression through the cell cycle. Prostaglandin E1 and PGE2 have prolonged or residual effects on melanoma cells. Cells treated for 5 or 24 hours with 10 micrograms/ml PGE1 or cells treated for 8 or 24 hours with 10 micrograms/ml PGE2 demonstrated decreased proliferation and increased tyrosinase activity for 48 hours after removal of the PGs. The effects of PGs on the cell cycle were investigated by determining total DNA content in cells stained with propidium iodide (PI) and analyzed by a fluorescence activated cell sorter (FACS). Prostaglandin E1 blocked cells in G2 phase after 5 hours of treatment, corresponding to when inhibition of proliferation was first evident. Similarly, after 9 hours of treatment with PGE2, more cells were in late S, early G2 phase and less in G1 than their control counterparts. Also, melanoma cells were pulse-labeled with 5-bromo-2'-deoxyuridine (BrdUrd) prior to or at the end of PG treatment and then stained with a fluoresceinated monoclonal antibody to BrdUrd, and with PI. This allows one to observe how BrdUrd-labeled S-phase cells cycle with time. Both PGE1 and PGE2 inhibit proliferation by blocking cells in G2 phase of the cell cycle. The PG-induced block in G2 may be required by melanoma cells to synthesize mRNA and proteins that are essential for stimulation of tyrosinase activity. Ultrastructurally, only a subpopulation of the cells treated with PGE1 or PGE2 contained more mature melanosomes than control cells.     

Cell cycle kinetic measurements in an irradiated rat rhabdomyosarcoma using a monoclonal antibody to bromodeoxyuridine

Cell cycle kinetics after X-irradiation were studied in a solid rat rhabdomyosarcoma using a monoclonal antibody to bromodeoxyuridine (BrdUrd) in cells in which the DNA was labeled by BrdUrd. It could be shown that this tumor was composed of about 80% diploid host cells, and only 20% of the cells in the dissociated tumor were actually tetraploid tumor cells. When rats were injected intraperitoneally with BrdUrd to label S-phase cells in the tumor, only a fraction of both types of cells became labeled with BrdUrd during S-phase, even 24 h after injection. The diploid BrdUrd-labeled cells progressed rapidly into cycle; 4 h after injection of BrdUrd, labeled diploid G1-phase cells could be observed. Only 25% of the tetraploid S-phase cells could be labeled by a single injection of BrdUrd (160 mg/kg body weight). These labeled tetraploid cells progressed through the cell cycle with similar velocities as did labeled diploid cells. Using a "Mini Osmotic Pump" containing bromodeoxycytidine (BrdCyd) at high concentration (0.3 mol/L) that released BrdCyd continuously into the organism where it was converted to BrdUrd, it could be shown that after 2 days about 60% of cells in S-phase and 70% of cells in G2-phase were labeled. The fraction of labeled G2-phase cells in irradiated tumors (D = 10 and 20 Gy) was enhanced between 10 and 50 h after irradiation due to a radiation-induced G2 block in cycling tetraploid tumor cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

In situ DNA-RNA hybridization using in vivo bromodeoxyuridine-labeled DNA probe

An in vivo 5'-bromodeoxyuridine (BrdUrd) labeled DNA probe was used for in situ DNA-RNA hybridization. BrdUrd was incorporated into plasmid DNA by inoculating E. coli with Luria-Bertani (LB) culture medium containing 500 mg/L of BrdUrd. After purification of the plasmid DNA, specific probes of the defined DNA fragments, which contained the cloned insert and short stretches of the vector DNA, were generated by restriction endonuclease. The enzymatic digestion pattern of the BrdUrd-labeled plasmid DNA was the same as that of the non-labeled one. BrdUrd was incorporated in 15%-20% of the total DNA, that is, about 80% of the thymidine was replaced by BrdUrd. Picogram amounts of the BrdUrd-labeled DNA probe itself and the target DNA were detectable on nitrocellulose filters in dot-blot spot and hybridization experiments using a peroxidase/diaminobenzidine combination. The BrdUrd-labeled DNA probe was efficiently hybridized with both single stranded DNA on nitrocellulose filters and cellular mRNA in in situ hybridization experiments. Through the reaction with BrdUrd in single stranded tails, hybridized probes were clearly detectable with fluorescent microscopy using a FITC-conjugated monoclonal anti-BrdUrd antibody. The in vivo labeling method did not require nick translation steps or in vitro DNA polymerase reactions. Sensitive, stable and efficient DNA probes were easily obtainable with this method.     

BrdUrd labeling of S-phase cells in testes and small intestine of mice, using microwave irradiation for immunogold-silver staining: an immunocytochemical study.

In this study, BrdUrd labeling of S-phase cells in the small intestine and testes was accomplished using microwave irradiation. In this way crypt cells, spermatogonia, and Leydig cells could be labeled using removable plastic-embedded sections and immunogold-silver staining (IGSS). By using short periods of microwave irradiation for incubation of the monoclonal antibodies and the protein A-colloidal gold solution, the detection of BrdUrd-labeled cells could be remarkably enhanced. A comparative study of BrdUrd labeled spermatogonia in the testis of a Cpb-N mouse that received both [3H]-thymidine and BrdUrd proved that 90% of the BrdUrd-labeled cells also showed [3H]-thymidine labeling. The radioactive [3H]-thymidine labeling was a time-consuming method of 4 weeks' duration, whereas the BrdUrd-labeled cells could be labeled, fixed, enhanced, and counterstained in less than 3 hr. This investigation proves that BrdUrd labeling of S-phase cells can be a reliable, reproductive, rapid, and non-radioactive alternative method for [3H]-thymidine labeling of proliferating cells.     

In situ DNA-RNA hybridization using in vivo bromodeoxyuridine-labeled DNA probe

Summary An in vivo 5-bromodeoxyuridine (BrdUrd) labeled DNA probe was used for in situ DNA-RNA hybridization. BrdUrd was incorporated into plasmid DNA by inoculating E. coli with Luria-Bertani (LB) culture medium containing 500 mg/L of BrdUrd. After purification of the plasmid DNA, specific probes of the defined DNA fragments, which contained the cloned insert and short stretches of the vector DNA, were generated by restriction endonuclease. The enzymatic digestion pattern of the BrdUrd-labeled plasmid DNA was the same as that of the non-labeled one. BrdUrd was incorporated in 15%–20% of the total DNA, that is, about 80% of the thymidine was replaced by BrdUrd. Picogram amounts of the BrdUrd-labeled DNA probe itself and the target DNA were detectable on nitrocellulose filters in dot-blot spot and hybridization experiments using a peroxidase/diaminobenzidine combination. The BrdUrd-labeled DNA probe was efficiently hybridized with both single stranded DNA on nitrocellulose filters and cellular mRNA in in situ hybridization experiments. Through the reaction with BrdUrd in single stranded tails, hybridized probes were clearly detectable with fluorescent microscopy using a FITC-conjugated monoclonal anti-BrdUrd antibody. The in vivo labeling method did not require nick translation steps or in vitro DNA polymerase reactions. Sensitive, stable and efficient DNA probes were easily obtainable with this method.     

Cell cycle analysis using the monoclonal antibody Ki-67

We determined by flow cytometry the proportion of cells in cycle with the monoclonal antibody Ki-67 and also in S-phase after incorporation of bromodeoxyuridine (BrdUrd) with monoclonal antibody to BrdUrd. The monoclonal antibody Ki-67 was useful to detect a nuclear antigen present only in proliferating cells but not expressed in resting (Go) cells. Cell preparation to measure BrdUrd amount incorporated into cellular DNA was difficult but this anti-BrdUrd antibody was useful for measuring the rate of DNA synthesis and for the analysis of precious cell kinetics. These antibodies may provide useful information of cell kinetics.     

Cell kinetic studies of in situ human brain tumors with bromodeoxyuridine

At the time of surgery, 18 patients with various brain tumors were given a 1-h i.v. infusion of bromodeoxyuridine (BrdUrd), 150-200 mg/m2. At an infusion rate of 200 mg/m2/h, serum BrdUrd levels of 8 microM were achieved. After the infusion, tumor tissue was obtained and divided into two portions. One portion was fixed in 70% ethanol, embedded in paraffin, and sectioned; the sections were deparaffinized, denatured with 2 N HCl, and reacted with monoclonal antibodies against BrdUrd (anti-BrdUrd MAb). BrdUrd-labeled nuclei were demonstrated satisfactorily by an indirect peroxidase method. The other portion was dissociated into single cells with a DNase enzyme cocktail and reacted with FITC-conjugated anti-BrdUrd MAb to determine the percentage of BrdUrd-labeled cells or with chromomycin A3 for DNA analysis. The single-cell suspensions were analyzed by flow cytometry. The fraction of S-phase cells in the tissue sections was similar to both the percentage of BrdUrd-labeled nuclei and the S-phase fraction determined by flow cytometric analysis. The results obtained with BrdUrd-labeled nuclei were similar to those obtained from previous autoradiographic studies of various brain tumors exposed to a pulse of 3H-thymidine. Since BrdUrd is not radioactive and is nontoxic at the dosage used, these techniques, together with the histopathological diagnosis, may help to predict the biological malignancy of individual tumors.     

Fluorescence enhancement of DNA-bound TO-PRO-3 by incorporation of bromodeoxyuridine to monitor cell cycle kinetics.

BACKGROUND:The detection of DNA-incorporated bromodeoxyuridine (BrdUrd) in mammalian cells is a well-known and important technique to study cell cycle. The use of TO-PRO-3 for detection of BrdUrd substitution of DNA by dual-laser flow cytometry has been investigated. METHODS:Fluorescence enhancement of TO-PRO-3 in BrdUrd-labeled cells is registered in combination with the fluorescence emission of the intercalating dye propidium iodide (PI) as a total DNA stain to give bivariate DNA/BrdUrd histograms. By the low concentration of only 0.3 mircoM TO-PRO-3, BrdUrd detection is optimized, and undisturbed total DNA content by PI can be detected as well. TO-PRO-3 is excited by a red HeNe laser and PI by an argon ion laser. RESULTS:In order to understand the binding of TO-PRO-3, energy transfer from PI to TO-PRO-3 has been measured as well as the influence of an external DNA binding dye such as Hoechst 33258 with Adenine-Thymine (AT) binding specificity. Cell cycle studies of human SCL-2 keratinocytes and mouse 3T3 cells prove the method to be as generally applicable as the classical BrdUrd/Hoechst quenching technique, but without need for expensive ultraviolet laser excitation. No BrdUrd sensitivity could be found for the similar dyes TO-PRO-1 and YO-PRO-3, whereas TO-PRO-5 and YOYO-3 showed only very little sensitivity to BrdUrd labeling as compared with TO-PRO-3. CONCLUSIONS:Cell cycle studies of mammalian cells can be done by dual-laser flow cytometry without the need for ultraviolet lasers by using the BrdUrd-dependent fluorescence enhancement of TO-PRO-3. Total DNA content can be measured simultaneously using PI.     

B cell stimulatory factor-1 (interleukin 4) activates macrophages for increased tumoricidal activity and expression of Ia antigens

Macrophages are activated by lymphokines (LK) to kill tumor cell and microbial targets. Interferon-gamma (IFN) is the major LK activity in conventional, antigen or mitogen-stimulated spleen cell culture fluids for induction of these macrophage effector functions. In view of the recent demonstration that murine macrophage-like cell lines have receptors for B cell stimulatory factor-1/interleukin 4 (BSF-1), a possible role for BSF-1 in regulation of macrophage function was considered. In this communication, thioglycollate-elicited murine peritoneal macrophages were shown to express about 2300 high affinity (Ka approximately 2 X 10(10) M-1) BSF-1 receptors/cell. Peritoneal macrophages treated with purified, T cell-derived BSF-1 developed potent tumoricidal activity against fibrosarcoma target cells. The concentration of BSF-1 that induced 50% of maximal tumor cytotoxicity was 38 +/- 4 U/ml for seven experiments; similar dose-responses were observed with recombinant BSF-1. That BSF-1 dose-responses for induction of macrophage-mediated tumor cytotoxicity were not affected by 5 micrograms/ml polymyxin B suggested that contaminant endotoxins played little or no role in cytotoxic activity. BSF-1 alone (less than or equal to 500 U/ml) was not directly toxic to tumor cells or macrophages. Macrophage tumoricidal activity induced by BSF-1 but not by IFN was inhibited greater than or equal to 90% with monoclonal anti-BSF-1 antibody. BSF-1 induced Ia antigen expression on peritoneal macrophages and increased (twofold to threefold) FcR(II)-dependent binding of murine IgG immune complexes to bone marrow-derived macrophages (greater than 98% macrophages). Based on these findings, it was concluded that BSF-1 is a potent macrophage activation factor.     

A novel rat monoclonal antibody directed against a population of activated mouse peritoneal macrophages

A hybridoma clone secreting rat monoclonal antibody (MAB) designated as 3F3.5F and which reacted with a population of activated tumoricidal mouse peritoneal macrophage (M phi) was produced by the fusion of mouse myeloma cells with rat spleen cells immunized against adherent BCG-activated mouse peritoneal exudate cells (adherent BCG-PEC). The antibody was cytotoxic and of the rat IgM class. The specific reactivity of the antibody with mouse primary cells and cell lines was examined by complement-dependent cytotoxicity and indirect immunofluorescence flow cytometry analysis. The antibody was found to bind to about 40% of the adherent BCG-PEC activated in vivo and elicited peritoneal macrophages activated in vitro by lymphokine and lipopolysaccharide (LPS), to about 35% of polymorphonuclear neutrophils (PMN) 15 hr after intraperitoneal injection of BCG, to about 30% of bone marrow cells from BCG-infected mice, to about 10% of P815 mastocytoma cells and to thioglycollate-induced PEC to some degree. It did not bind to other cells tested including BCG-induced peritoneal lymphocytes, non-tumoricidal PEC, thymocytes, spleen cells, resting bone marrow cells from normal mice, lymphomas, myelomas, fibroblasts, or macrophage-cell lines. Pretreatment of adherent BCG-PEC with MAB 3F3.5F and rabbit complement caused a considerable decrease in tumor cytotoxicity toward P815 cells, but the same pretreatment of non-adherent BCG-PEC had no inhibitory effect on natural killer activity for YAC-1 cells.     

A monoclonal antibody to a differentiation antigen present on mature human macrophages and absent from monocytes

A monoclonal antibody is described that has been generated in the mouse against cultured human blood monocytes/macrophages. The antibody, designated 25F9, belongs to the IgG1 subclass, detects antigens of m.w. 86,000, and does not react with freshly isolated blood monocytes but reacts with monocytes after 3 days of culture. The expression of the 25F9 antigen on macrophages increases with culture time. Furthermore, the antibody is negative on platelets, granulocytes, lymphocytes, and a large number of human cell lines except the two melanoma lines MeWo and Mel 57. In cryostat sections of normal human tissue (skin, lung, liver, thymus) and of inflammatory or neoplastic tissue (cutaneous lymphoma, eczema, BCG-granuloma, and melanoma), the antibody reacts with scattered macrophages in the dermis but not with epidermal Langerhans cells, with alveolar macrophages, with liver Kupffer cells, and with scattered macrophages in the cortex and medulla of thymus. In eczema, BCG-granuloma, and cutaneous lymphoma, only a few infiltrating macrophages were stained. On the other hand, a large number of macrophages and melanophages reacted positively in melanoma. In some cases melanoma cells also stained weakly positive. Thus, the antibody detects a differentiation antigen preferentially expressed on mature, tissue-fixed macrophages and absent from blood monocytes.     

Isolation and characterization of monoclonal antibodies reactive with endothelial cells

Monoclonal antibodies were generated to antigens on cultured human umbilical vein endothelial cells. Spleen cells from BALB/c mice, immunized with low passage cultures of human umbilical vein endothelial cells, were fused with the non-secretory myeloma line, P3 x 63Ag 8.653. Hybridoma supernatants were screened for the desired immunological reactivity using ELISA binding assays. Hybridomas secreting antibodies reacting with the immunizing endothelial cells, but not with peripheral blood mononuclear cells, were cloned by limiting dilution and three stable clones were chosen for study. Further testing by ELISA revealed that each antibody displayed a unique pattern of reactivity. One antibody, 14E5, reacted with the macrophage-like cell line DHL-2, cultured macrophages derived from peripheral blood monocytes, and macrophages derived from malignant effusions. The antibody failed to react with fibroblasts or bovine endothelial cells. The second antibody, 12C6, reacted with human and primate fibroblasts and endothelial cells derived from bovine arteries, but not with mature macrophages. The third clone, 10B9, reacted only with the immunizing endothelial cells and the immature-macrophage line U-937. All three antibodies failed to react with long-term human B or T lymphoblastoid cell lines, leukemic cell lines, or murine macrophage lines. None of the antibodies reacted with a battery of human epithelial derived cell lines or primary cultures of human epithelial cells. Indirect immunofluorescence assays revealed that the antigens were expressed on the cell surface. These antibodies should prove useful as differentiation markers of human endothelial cells and in studies of endothelial cell function.     

Cytokinetic analysis of lung cancer by bromodeoxyuridine labeling of cytology specimens

Recent flow cytometric (FCM) studies have indicated the prognostic value of S-phase cells (SPF) in lung cancer. More refined cytokinetic analysis can be obtained by dual-parameter FCM, labeling S-phase cells with 5-bromodeoxyuridine (BrdUrd), which can be detected using a monoclonal antibody (MoAb) to BrdUrd. Tumor cells obtained through bronchoscopic brush were incubated for 1 hr in RPMI 1640 medium with 10% fetal calf serum and 10 microM BrdUrd. After fixation in ethanol, pepsin treatment, and DNA denaturation, the nuclei were stained with anti-BrdUrd MoAb and propidium iodide. From 14 of 20 patients, sufficient material was obtained (three adenocarcinoma and seven squamous cell, one giant cell, and three small cell carcinoma). The measured SPF ranged from 5.2% to 26%. The labeling index (LI), calculated as the ratio of the number of BrdUrd-labeled cells to the total number of aneuploid cells, or diploid cells in the case of a diploid tumor, ranged from 1.2% to 16.7%; LI and SPF correlated significantly (r = 0.69). In this study, we have demonstrated the feasibility of determining the actively DNA-synthesizing cells on brush material from lung cancer cells. In addition, some extra information can be obtained about the SPF population, including the fraction of unlabeled SPF, which could be of prognostic significance.     

Influence of incorporated 5-bromodeoxyuridine on the frequencies of spontaneous and induced sister-chromatid exchanges, detected by immunological methods

We have utilized monoclonal antibody against BrdUrd to detect sister-chromatid exchanges in CHO cells. This technique allows detection of SCEs at very low levels of BrdUrd incorporation. At incorporation level of 0.5%, a frequency of about 2 SCEs/cell/cycle was found. In a UV-sensitive mutant (43-3B) which has an increased spontaneous frequency of SCEs, it is found that this increase is due to incorporated BrdUrd. In MMS- and MMC-treated cells, an influence of BrdUrd on the frequencies of induced SCEs was found only when high concentrations of mutagens were employed.     

Release of hemopoietic factors by normal human T cell lines with either suppressor or helper activity

We analyzed the release of activities capable of stimulating the in vitro growth of human hemopoietic progenitor cells by long-term cultured T cell growth factor (TCGF)-dependent human T lymphocytes. Seven cell lines tested produced colony-stimulating activity (CSA) as well as burst-promoting activity (BPA). The CSA stimulated primarily the growth of the cells forming colonies after 14 days of incubation. In addition the supernatants from these seven T-cell lines showed the ability to induce the in vitro growth of mixed granulocyte, erythroid, megakaryocyte, macrophage colonies (CFU-GEMM). The release of hemopoietic factors did not depend on the presence of accessory cells or phytohemagglutinin or serum during the incubation for factor production. In six of the T cell lines the majority of the cells were reactive to the OKT 8 monoclonal antibody (MoAb), whereas one cell line contained mostly OKT 4+ cells. Suppressor activity was detected in three tested OKT 8+ cell lines, while the one OKT 4+ displayed helper activity. All cell lines produced hemopoietic factors with equal efficiency. These results indicate that factors affecting human hematopoiesis are produced by normal T lymphocytes in long-term culture and this property is not related to the helper or suppressor activity of the cultured cells.     

Macrophage colony-stimulating factor enhances monocyte and macrophage antibody-dependent cell-mediated cytotoxicity

In vitro culture of either human peripheral blood monocytes or murine peritoneal macrophages for 72 hr in the presence of macrophage colony-stimulating factor (M-CSF) dramatically increased their subsequent ability to mediate antibody-dependent cellular cytotoxicity (ADCC). The M-CSF-treated cells were more effective in ADCC at lower effector to target cell ratios and in the presence of lower concentrations of tumor-specific monoclonal antibody than the untreated control cells. Two other hematopoietic cytokines, granulocyte-macrophage colony-stimulating factor and interleukin-3, reported to enhance other macrophage effector functions were ineffective in promoting the development of ADCC by cultured human monocytes. All three hematopoietic growth factors were capable of enhancing the ability of the cultured monocytes to secrete TNF alpha; however, TNF alpha is unlikely to be an important cytotoxic factor in ADCC because neutralizing antibodies against TNF alpha had no affect on ADCC in vitro. Further, much higher concentrations of M-CSF were required to augment monocyte TNF alpha release (20-100 ng/ml) than ADCC capacity (1-10 ng/ml). These results suggest that M-CSF administration might prove effective in increasing the tumoricidal activities of tumor-specific monoclonal antibodies by enhancing the capacity of monocytes and macrophages to mediate ADCC.     

Cell kinetics in mouse epidermis studied by bivariate DNA/bromodeoxyuridine and DNA/keratin flow cytometry.

Hairless mice were injected intraperitoneally with bromodeoxyuridine (Brd-Urd). Basal cells were isolated from epidermis, fixed in 70% ethanol, and prepared for bivariate BrdUrd/DNA flow cytometric (FCM) analysis. Optimum detection of incorporated BrdUrd in DNA was obtained by combining pepsin digestion and acid denaturation. The cell loss was reduced to a minimum by using phosphate-buffered saline containing Ca2+ and Mg2+ to neutralize the acid. The percentage of cells in S phase and the average uptake of BrdUrd per labelled cell in eight consecutive windows throughout the S phase were measured after pulse labelling at intervals during a 24 h period. Furthermore, the cell cycle progression of a pulse-labelled cohort of cells was followed up to 96 h after BrdUrd injection. In general the results from both experiments were in good agreement with previous data from 3H-thymidine labelling studies. The percentage of cells in S phase was highest at night and lowest in the afternoon, whereas the average uptake of BrdUrd per labelled cell showed only minor circadian variations. There were no indications that BrdUrd significantly perturbed normal epidermal growth kinetics. A cell cycle time of about 36 h was observed for the labelled cohort. Indications of heterogeneity in traverse through G1 phase were found, and the existence of slowly cycling or temporarily resting cells in G2 phase was confirmed. There was, however, no evidence of a significant population of temporarily resting cells in the S phase. Bivariate DNA/keratin FCM analysis revealed a high purity of basal cells in the suspensions and indicated that the synthesis of the differentiation-keratin K10 was turned on only in G1 phase and after the last division.