Effects of strength training on muscle cellular outcomes in prostate cancer patients on androgen deprivation therapy.

Androgen deprivation therapy (ADT) improves life expectancy in prostate cancer (PCa) patients, but is associated with adverse effects on muscle mass. Here, we investigated the effects of strength training during ADT on muscle fiber cross-sectional area (CSA) and regulators of muscle mass. PCa patients on ADT were randomized to 16 weeks of strength training (STG) (n = 12) or a control group (CG; n = 11). Muscle biopsies were obtained from m. vastus lateralis and analyzed by immunohistochemistry and western blot. Muscle fiber CSA increased with strength training (898 µm(2) , P = 0.04), with the only significant increase observed in type II fibers (1076 µm(2) , P = 0.03). There was a trend toward a difference in mean change between groups myonuclei number (0.33 nuclei/fiber, P = 0.06), with the only significant increase observed in type I fibers, which decreased the myonuclear domain size of type I fibers (P = 0.05). Satellite cell numbers and the content of androgen receptor and myostatin remained unchanged. Sixteen weeks of strength training during ADT increased type II fiber CSA and reduced myonuclear domain in type I fibers in PCa patients. The increased number of satellite cells normally seen following strength training was not observed.

Intricacies of redoxome function demonstrated with a simple in vitro chemiluminescence method, with special reference to vitamin B12 as antioxidant.

The homeostatic control of the redox system (the redoxome) in mammalian cells depends upon a large number of interacting molecules, which tend to buffer the electronegativity of cells against oxidants or reductants. Some of these components kill - at high concentration - microbes and by-stander normal cells, elaborated by professional phagocytes. We examined whether a simple, in vitro chemiluminescence set-up, utilizing redox components from human polymorphonuclear neutrophils (PMN) and red blood cells (RBC), could clarify some unexplained workings of the redoxome. PMN or purified myeloperoxidase (MPO) triggers formation of reactive oxygen species (ROS), quantified by light emission from oxidized luminol. Both PMN and RBC can generate abundant amounts of ROS, necessitating the presence of a high-capacity redoxome to keep the cellular electronegativity within physiological limits. We obtained proof-of-principle evidence that our assay could assess redox effects, but also demonstrated the intricacies of redox reactions. Simple dose-responses were found, as for the PMN proteins S100A9 (A9) and S100A8 (A8), and the system also revealed the reducing capacity of vitamin B12 (Cbl) and lutein. However, increased concentrations of oxidants in the assay mixture could decrease the chemiluminescence. Even more remarkable, A9 and NaOCl together stimulated the MPO response, but alone they inhibited MPO chemiluminescence. Biphasic responses were also recorded for some dose-response set-ups and are tentatively explained by a 'balance hypothesis' for the redoxome.

C-reactive protein triggers calcium signalling in human neutrophilic granulocytes via FcγRIIa in an allele-specific way.

C-reactive protein (CRP) binds to Fcγ-receptors, FcγRIIa (CD32) with high affinity and to FcγRIa (CD64) with low affinity. The binding to CD32 has been shown to be allele specific, that is, it binds to R/R131 but not to H/H131. Little is known about the cooperation of CRP and neutrophilic granulocytes (PMNs) in inflammatory reactions. The purpose of the present study was to examine CRP signalling in human PMNs, and whether this signalling is also allele specific. Cytosolic calcium of PMN was measured in a single-cell digital imaging system. Receptor expression and polymorphism were studied by real-time RT-PCR, flow cytometry and standard PCR. C-reactive protein induced cytosolic calcium signals in PMNs from homozygote R/R131 donors, but not in PMNs from heterozygote R/H131 donors. However, after the heterozygote PMNs had been incubated with IFN-γ (100 U/ml) for 2 h, both the proportion of cells responding and the size of the CRP-induced calcium signals increased. IFN-γ increased mRNA expression of CD64 about fivefold and surface protein expression of CD64 about fourfold. The calcium signal elicited by CRP was augmented by PMN adhesion to fibronectin, but almost totally abrogated by sphingosine kinase inhibitors. The signals were partly dependent on calcium influx. In conclusion, calcium signalling instigated by CRP in human PMN is FcγRIIa allele specific, as R/R131 responded to CRP, whereas R/H131 did not. However, increased expression of FcγRIa (CD64), stimulated by IFN-γ, can augment calcium signalling by CRP in low-responders. This suggests that the state of the PMNs, as well as the genetic origin, affect sensitivity for CRP.

A COX-2 inhibitor reduces muscle soreness, but does not influence recovery and adaptation after eccentric exercise.

The aim of this study was to investigate the effect of a cyclooxygenase (COX)-2 inhibitor on the recovery of muscle function, inflammation, regeneration after, and adaptation to, unaccustomed eccentric exercise. Thirty-three young males and females participated in a double-blind, placebo-controlled experiment. Seventy unilateral, voluntary, maximal eccentric actions with the elbow flexors were performed twice (bouts 1 and 2) with the same arm, separated by 3 weeks. The test group participants were administered 400 mg/day of celecoxib for 9 days after bout 1. After both bouts 1 and 2, concentric and isometric force-generating capacity was immediately reduced (approximately 40-50%), followed by the later appearance of muscle soreness and increased serum creatine kinase levels. Radiolabelled autologous leukocytes (detected by scintigraphy) and monocytes/macrophages (histology) accumulated in the exercised muscles, simultaneously with increased satellite cell activity. These responses were reduced and recovery was faster after bout 2 than 1, demonstrating a repeated-bout effect. No differences between the celecoxib and placebo groups were detected, except for muscle soreness, which was attenuated by celecoxib. In summary, celecoxib, a COX-2 inhibitor, did not detectably affect recovery of muscle function or markers of inflammation and regeneration after unaccustomed eccentric exercise, nor did the drug influence the repeated-bout effect. However, it alleviated muscle soreness.

Native Whey Induces Similar Post Exercise Muscle Anabolic Responses as Regular Whey, Despite Greater Leucinemia, in Elderly Individuals.

OBJECTIVE: Elderly muscle seems less sensitive to the anabolic stimulus of a meal. Changes in blood concentrations of leucine are suggested as one important trigger of the anabolic response in muscle. The aim of this study was to investigate whether native whey protein, containing high amounts of leucine, may be a more potent stimulator of muscle protein synthesis (MPS) in elderly than regular whey protein (WPC-80) or milk. DESIGN: Randomized controlled partial crossover. SETTING: Norwegian School of Sport Sciences. PARTICIPANTS: 21 healthy elderly men and women (≥70 years). INTERVENTION: Participants received either 20 g of WPC-80 and native whey (n = 11) on separate days in a crossover design, or milk (n = 10). Supplements were ingested immediately and two hours after a bout of lower body heavy-load resistance exercise. MEASUREMENTS: Blood samples and muscle biopsies were collected to measure blood concentrations of amino acids by gas-chromatography mass spectrometry (GCMS), phosphorylation of p70S6K, 4E-BP1 and eEF-2 by immunoblotting and mixed muscle fractional synthetic rate (FSR) by use of [2H5]phenylalanine-infusion, GCMS and isotope-ratio mass spectrometry. RESULTS: Native whey increased blood leucine concentrations more than WPC-80 (P < 0.05), but not p70S6K phosphorylation or mixed muscle FSR. Both whey supplements increased blood leucine concentrations (P < 0.01) and P70S6K phosphorylation more than milk (P = 0.014). Native whey reached higher mixed muscle FSR values than milk (P = 0.026) 1-3h after exercise. CONCLUSIONS: Despite greater increases in blood leucine concentrations than WPC-80 and milk, native whey was only superior to milk concerning increases in MPS and phosphorylation of P70S6K during a 5-hour post-exercise period in elderly individuals.

Inhibitors of angiogenesis selectively reduce the malignant cell load in rodent models of human myeloid leukemias.

Angiogenesis is essential for growth and metastasis of solid tumors and probably also for hematological malignancies. Angiogenic inhibitors, like endostatin (ES) and PI-88, retard cancer growth. We tested these in mice with juvenile myelomonocytic leukemia (JMML), and in rats with acute myeloid leukemia (BNML). Eight weeks after transplantation and with a continuous drug treatment for the last 4 weeks, the leukemic cell mass decreased from almost 90% of all bone marrow cells to about 15 and 45% with ES, to about 35 and 55% with PI-88, and to about 10 and 25% with ES + PI-88 in the leukemic mice and rats, respectively. The numbers of normal human bone marrow cells transplanted into mice were unchanged by the treatments. The microvessel density in leukemic animals given ES or PI-88 was 10-50% of that in untreated animals. Notably, simultaneous treatment with ES and PI-88 led to a reduction of about 95% in JMML mice and 85% in BNML rats. In vitro proliferation of either JMML or BNML cells was not significantly altered by either drug, demonstrating the selectivity of ES and PI-88 as angiogenic inhibitors. In conclusion, anti-angiogenic therapy may be a valuable adjunct to conventional treatment of leukemia.

Hydroxyurea (HU) in experimental hematology. II. Similarities and dissimilarities between HU and 3H-thymidine killing.

The lethal effects of hydroxyurea (HU) and 3H-thymidine (3H-dT) on mouse hematopoietic cells were compared after various experimental procedures. The aim was to explore the relative efficiency of these two methods in analyzing the kinetic properties of progenitor cells. Both methods indicated that 40-50% of progenitor cells assayed with diffusion chamber culture (DCPC) were in S phase 3 days after cyclophosphamide treatment. Effects of HU, but not 3H-dT, were altered by neostigmine triggering of normal DCPC into cell cycle. On the other hand, cooling marrow cells before exposure to HU or 3H-dT largely abrogated the effect of HU, but not of 3H-dT. Blood-borne DCPC were not in cycle according to the HU effect. Separated blood DCPC were apparently in cycle, as judged with 3H-dT, but the Isopaque-Ficoll separation procedure rendered normal marrow DCPC susceptible to 3H-dT killing. When marrow cells were cultured in DC the HU technique appeared to be suitable for evaluation of modulation of progenitor cell (CFU-S or CFU-C) proliferation, whereas our previous experiments have shown that the 3H-dT technique is a convenient method to assess the initial triggering of CFU-S into cycle in DC culture.

How should we estimate confidence intervals for a ratio, for example, between counts of colony-forming cells?

When calculating a ratio, of colony counts, for example, experimental hematologists often provide no indication of the uncertainty of the estimates, or the uncertainty is computed using a method with partially unknown properties. We have used empirical data on survival of colony-forming cells after exposure to hydroxyurea to select assumptions regarding the statistical properties of the survival ratios. Then, we have evaluated four alternative methods for constructing confidence intervals for such ratios. With simulations, the adequacy of the methods is shown to depend on the assumed statistical distributions of the data and on the number of observations in the numerator and denominator. Especially with few observations, some methods may produce misleading results. An easily calculable confidence interval, formally based on lognormal distributions, seems to be applicable to a fairly wide range of other plausible statistical distributions as well; it may be a reasonable choice in many situations. Whatever method is chosen, the confidence intervals for ratios are wide, and more data than is generally appreciated are needed for a precise determination of a ratio.

Diffusion chamber culturing of haematopoietic cells: methodological investigations and improvement of the technique.

Mouse bone marrow cells were cultured to determine some basic characteristics of the diffusion chamber (DC) technique. The main findings were as follows: (i) Incorporated 3H-thymidine was conveniently measured after deposition of a cell sample on glass fibre discs followed by methanol washing. Varying the specific activity from approximately 2-approximately 20 Ci/mmole did not affect the relationship between DC cellularity and isotope incorporation. Isotope uptake was similar regardless of whether 3H-thymidine of high or low specific activity had been used. (ii) Higher cell yields were obtained when Millipore or Acropor filters were heat-sealed rather than glued to the plastic rings, when Millipore filters were moistened before DC filling, when we omitted gluing the closing plugs, and when we used an average pore size of 0.22 mum for the DC walls rather than one of 0.10 mum or 0.45 mum. Varying the ring thickness from 2.0-2.5 mm did not impair the cell growth, nor did removal of a softening agent from the plastic rings improve it. (iii) More cells were retrieved from DC carried by young rather than by older mice. Results were not influenced by sex or strain differences between the donor and the host or by the number of implants, whether single or double, within an individual host. (iv) Adding Ficoll to the pronase solution increased the yield of viable CFU-C. (V) Diffusion rate through the DC walls declined with increasing period of culturing, so that i.p. 3H-thymidine is not a flash label for 7-day cultures, for example. The great variability of 3H-thymidine diffusion into i.p. DC was markedly reduced by in vitro exposure to the unopened DC to the isotope. (vi) DC could be incubated in vitro in a medium devoid of protein for at least 6 hours without a fall in 3H-thymidine incorporation rate or CFU-C content, provided that pH was kept constant.

Thymic hormones and syngeneic T-lymphocytes are not required for leukopoiesis in an in vivo culture system for mouse bone marrow cells.

In order to explore the regulatory influence of thymic hormones and T cells on leukopoiesis , bone marrow cells from normal and athymic nude mice were cultured in peritoneal diffusion chambers (DC) that were implanted intraperitoneally into either normal or nude mice. T-cell-deficient nu/nu-C3H marrow cells formed slightly above normal numbers and nu/nu-BALB/c formed slightly below normal numbers of progenitor cells for granulocytes and/or macrophages in four-day DC cultures. Seven-day leukopoiesis in DC, as estimated by 3H-thymidine incorporation and differential cell counts, was not detectably affected by T-cell absence from nu/nu-BALB/c marrow. Leukopoiesis , including formation of progenitor cells, was not significantly different in normal and T-cell-deficient DC hosts. Normal thymocytes added to the T-cell-deficient bone marrow inocula decreased the generation of progenitor cells. These results indicate that (a) expansion of a proliferating granulocyte-macrophage progenitor cell population takes place in in vivo DC cultures without the presence of thymic hormones and T-lymphocytes, and that (b) T-lymphocytes may either stimulate or inhibit progenitor cell growth, and the balance between stimulation and inhibition apparently varies between mouse strains.

Diffusion chamber (DC) culturing of haemopoietic cells: a reliable assay system for regulators of proliferation?

If i.p. DC culturing of haemopoietic cells shall serve as a reliable assay for systemic and/or local peritoneal regulators of proliferation, cell growth must not be restricted by the diffusion capacity through the chamber membranes. Diffusion was assessed by measuring uptake in DC or 51Cr-EDTA of 3H-thymidine, given intravenously to mouse hosts or to chambers incubated in vitro. Fractional incorporation of 3H-thymidine, numbers of mouse spleen colony-forming stem cells, agar colony-forming progenitors (of granulocytes and macrophages), and total cell numbers were taken to reflect cell growth. Changing the diffusion capacity did not lead to marked changes in cell growth; and vice versa, culturing cells under host or chamber conditions that increased cell growth was not associated with an appreciably increased diffusion capacity. Diffusion was increased by replacing Acropor with Millipore DC or by repeatedly cleaning the DC during the culture period. Cell growth was increased by using irradiated rather than normal host mice, and Nucleopore rather than Millipore DC in irradiated mice. Increasing the oxygen delivery to the cultured cells by using polycythaemic and hyperoxic hosts did not enhance cell growth either.

Flow cytometry of mouse bone marrow cells cultured in vivo or in vitro.

We have developed new methods for counting cyto-(fluoro)-metrically cells retrieved from diffusion chamber and methylcellulose cultures of mouse bone marrow. Total cell counting and DNA distribution measurements were based on detection of the fluorescence from propidium iodide stained nuclei. Staining of cell nuclei with propidium iodide further allowed the separate enumeration of avid phagocytes that had endocytosed fluorescent latex beads. Finally, we quantified peroxidase positive cells by using o-Dianisidine as a substrate for the enzyme, the formation of brown reaction product being enhanced by the presence of lidocaine and dichlorophenol. We found the new techniques precise, reliable and suitable for analysis of cell growth and maturation in culture.

The leukopoietic cytokine granulocyte colony-stimulating factor increases blood flow to rat bone marrow.

The current knowledge concerning the blood supply to hematopoietic bone marrow during increased marrow metabolism is scanty. We have previously shown that an accelerated erythropoiesis in the awake rat is accompanied by a rapid increase in perfusion of the tibial marrow and its bony encasement. We have now measured blood flow to tibial marrow and bone in rats with stimulated granulopoiesis, caused by injection of recombinant human granulocyte colony-stimulating factor (rhG-CSF). In awake, adult rats, blood flow was measured with the microsphere method before and at intervals during a 48-hour period after subcutaneous (s.c.) injection of rhG-CSF (10 micrograms/kg). Administration of rhG-CSF caused a marked leukocytosis, mostly due to an increase in blood granulocytes, amounting to 4 times the control value at 8 hours. Concomitantly, the perfusion of tibial marrow rose to about 200% of control by 8 hours before it declined toward baseline. Denervation of the marrow had no effect on this hyperemic response. The perfusion of tibial bone was apparently unaffected by rhG-CSF injection. We conclude that rhG-CSF injection increases blood flow to hematopoietic marrow, but not to bone. This may have important implications for marrow transplantation and drug therapy for patients with marrow failure.

Some methods of assaying inhibitors of cell proliferation.

Inhibition of cell proliferation by leucocyte extracts containing the granulocytic chalone has been monitored in various ways. Target cells were rat bone marrow cells and chloroleukaemia cells cultured on cover glasses, as well as mouse marrow cells cultured in vivo and in vitro in diffusion chambers. Provided that the technique is optimized, the in vitro rat cell assays yielded reliable results with different types of radioactive DNA precursors. With the diffusion chamber techniques cells synthesizing DNA were killed specifically by hydroxyurea, rather than labelling them with e.g. 3H-thymidine. Inhibition of proliferation then showed up as a decreased cell killing, as compared with the controls. Inhibition of proliferation of both progenitor and transit cells could be demonstrated in this way, without interference by added thymidine.

Endogenous nitric oxide causes vasodilation in rat bone marrow, bone, and spleen during accelerated hematopoiesis.

There is a marked increase in blood flow to rat bone marrow during increased erythro- or granulopoiesis. Furthermore, stimulated erythropoiesis increases bone and splenic perfusion, whereas granulopoietic hyperactivity does not. The mechanism behind this hyperemia is unknown. Endogenous nitric oxide (NO) has been shown to be a potent vasodilator in many vascular beds, but its possible role in the regulation of bone marrow, bone, and spleen vascular resistance and perfusion has not been explored. With the radioactive microsphere method, we determined blood flow to bone marrow, bone, and spleen in awake rats. Eight rats were bled heavily (1.5% of body weight), eight others received 10 micrograms/kg recombinant human granulocyte colony-stimulating factor (rhG-CSF) subcutaneously, and eight other untreated rats served as controls. We used 300 micrograms/kg, intraaortal, of the potent NO synthase blocker N-monomethyl-L-arginine (L-NMMA) (Calbiochem, La Jolla, CA). The inhibition of NO formation was subsequently reversed with 1000 mg/kg intraaortal arginine. Marrow vascular resistance was reduced to approximately 30% of control baseline in the experimental rats 10 hours after hematopoietic stimulation with either bleeding or rhG-CSF. Concomitantly, marrow blood flow increased to about 260% of control baseline in the bled rats, while it almost tripled after rhG-CSF injection. Inhibition of NO formation increased marrow vascular resistance in all three groups. After L-NMMA treatment, marrow perfusion was reduced to about 50% of baseline in the bled and 75% in the rhG-CSF-treated rats, while perfusion in the controls remained apparently unaltered. These changes were completely reversed with arginine. The increases in vascular resistance after NO blockade could not be explained by a concomitant change in arterial blood pressure. L-NMMA increased the vascular resistance in the bone and spleen both in controls and in stimulated rats, but since arterial blood pressure rose proportionally, perfusion remained unchanged. We conclude that NO plays an important role in the regulation of both the normal bone marrow vascular tone and the vasodilation that occurs during accelerated hematopoiesis. NO apparently also regulates bone and splenic vascular tone, but less conspicuously than in the stimulated bone marrow.

Hydroxyurea (HU) in experimental hematology. I. Characterization of in vitro and in vivo effects on progenitor and transit cells.

The lethal effect of hydroxyurea (HU) on normal and regenerating mouse bone marrow was examined in vivo and in vitro. Progenitor cells (DCPC) were assayed by their capacity to form granulocytes and macrophages in 7-day diffusion chamber (DC) cultures. Dose-response experiments showed that a plateau effect was obtained in vitro for HU concentrations above 1 mM, killing about 40% of regenerating progenitor cells or normal proliferative granulocytes. Similarly, a plateau effect was found for doses exceeding about 15 mg i.p., when lethal effects on 3-4 day DC cultures were measured. Time-response studies in vitro showed that about 60% of maximum killing was achieved after only 10 min, with a levelling off of the effect for exposure times between 1 and 3 h. Total depression of 3H-thymidine incorporation in DC cultures was achieved in less than 0.5 h, and subsided within 4-6 h, after one i.p. injection of 23 mg HU. HU had no appreciable effect on DCPC of adult normal marrow, but killed 15% of agar colony-forming cells.

Digital image analysis of hematopoietic colonies in vitro.

A system for automatic analysis of in vitro hematopoietic colonies is described and evaluated. With the standard resolution provided by video cameras, the improvement in visualization obtained using features other than size and darkness when classifying potential colonies appears to be limited. We confirmed this by comparing results obtained with the test system with those obtained with a commercial one. However, for some applications it may be useful to supplement the system with specific methods, e.g., to separate merged colonies. Digital image analyses provide new possibilities, for instance of measuring the total cellularity of the dish or analyzing colonies according to the size and cell density of each colony. Examples provided are time course studies of colony development, cellularity feedback effects on colony sizes, and bell-shaped dose-response curves for the growth stimulation obtained by certain conditioned media on a subpopulation of progenitor cells that gives rise to large colonies.

Simultaneous assessment of migration and proliferation of murine fibrosarcoma cells, as affected by hydroxyurea, vinblastine, cytochalasin B, Razoxane and interferon.

Using porous cell culture chambers, we have simultaneously assessed growth and locomotion of cancer cells to investigate whether certain agents affect cell motility in addition to cell division. First, cells from a murine fibrosarcoma cell line, 1.0/L1, were grown in ordinary flask cultures to determine appropriate cell inocula. Doses of agents were selected to reduce the final 4 day culture cellularity to about 50%, when present during the last two days of culturing. Secondly, the effects of these agents on cell numbers in the porous chambers and on cell migration out of the chambers ('emigration fraction') were recorded. We also examined, using a similar type of porous chamber, whether the agents could affect leucocyte chemotaxis. Hydroxyurea (an inhibitor of DNA synthesis) reduced cancer cell emigration as well as cell growth, without interfering with leucocyte chemotaxis. Cytochalasin B (a microfilament disrupting agent) inhibited cancer cell motility and growth, as well as leucocyte chemotaxis. Vinblastine (a microtubule disrupting agent), at the very low dose chosen, reduced cancer cell growth, but did not consistently affect the migration of either cell type. The experimental anti-metastasis agent Razoxane reduced growth, but had no detectable effects on motility. High doses of natural murine interferon-alpha/beta weakly inhibited both cancer cell growth and locomotion. This motivates for further studies of these and other cytokines, as treatment with agents inhibiting cancer cell locomotion might possibly prevent peri-operative spread of cancer in patients.

Modulation by interferons of human neutrophilic granulocyte migration.

The effect of various interferons (IFN) on neutrophilic granulocyte (PMN) random and directed migration is incompletely understood. We, therefore, investigated PMN migration with a novel micropore membrane technique. No chemotactic effect of either 10-10000 U/ml IFN-alpha or IFN-beta, or 1-1000 U/ml IFN-gamma was observed on PMN isolated from normal human venous blood. However, when present on both sides of the micropore membrane, all the IFN (1000 U/ml IFN-alpha and IFN-beta, 100 U/ml IFN-gamma) inhibited both random and directed migration toward zymosan-activated serum (ZAS). IFN-gamma was the most potent inhibitory agent and produced an inhibition of about 30%. When the bacterial peptide fMLP was used as a chemoattractant, IFN-gamma also depressed chemotaxis. Taking the reduced random migration of IFN-gamma treated cells into account, however, chemotaxis per se-toward both ZAS and fMLP-was not significantly affected. Random migration and directed migration assessed simultaneously with PMN from the same donor were clearly correlated for both control and IFN-gamma treated cells, suggesting that a general antimotility effect of IFN-gamma might explain both reduced random migration and chemotaxis. The antimotility effect of IFN-gamma was not dependent on protein synthesis or on tyrosine kinase activity. In fact, inhibition of tyrosine kinase with herbimycin A increased the ZAS-stimulated motility of both control and IFN-gamma-inhibited PMN. In conclusion, our data indicate that IFN depress both random and directed PMN migration by mechanisms that do not involve protein synthesis or protein tyrosine kinase activity.

A new assay for leukocyte chemotaxis using cell retrieval, electronic particle counting and flow cytometry.

A new micropore membrane assay for leukocyte migration has been devised. It permits the complete retrieval in monodisperse suspension of functionally intact cells that have traversed the membrane, thus allowing the application of precise, automated techniques, including flow cytometry and electronic particle counting. Hemocytometers may also be used. Direct comparison with 2 different conventional membrane methods showed that the new method performed superiorly. It was also much more economical with regard to time and labor. This technique permitted detection of functional differences between leukocytes isolated from blood in different ways. Data on the duration of concentration gradients in chemotaxis chambers are also presented.