Coulter Channelyzer Research Articles

Study of the possible repression for apoptosis in breast cancer vitrified cells (T-47D) in the presence of DMSO as a cryoprotectant

Correlated studies on volume distributions and cation (Na+ and K+) content of CHO cells in suspension were carried out after various exposures to hypertonic NaCl or sucrose (500–7550 mOsm in both the presence and absence of DMSO (5–20%; wv). The effects superimposed by ouabain (10−2–10−4 m), amphotericin B (6–18 μg/ml), and glutaraldehyde (1.25%) on the above-mentioned parameters were also investigated. Volumetric analysis of CHO cells with the Coulter Channelyzer indicated a biphasic dose-dependent response to hypertonic media, the duration of the a V, modal volume. b S, surviving fraction [data from Ref. (18)]. c Time dependency visible during a 2-hr observation period. shrinkage- re-expansion cycle being inversely proportional to medium osmolality. In hypertonic sucrose, CHO cells apparently fail to shrink up to approximately 1000 mOsm. In both hypertonic sucrose and NaCl there is a constant loss of intracellular K+ at velocities which depend primarily on medium tonicity and also on exposure length. K+ loss is accompanied by increased levels of intracellular Na+ only in cells exposed to hypertonic salt; in hypertonic (1–2 m) sucrose intracellular Na+ shows a progressive diminution. In NaCl-DMSO experiments the amount of K+ lost from the cells is less in comparison to that observed in “unprotected” cell suspensions. DMSO also mitigates excessive intracellular accumulations of Na+. In contrast to ouabain and amphotericin B, DMSO was found to slow down considerably the early volume re-expansion phase. Glutaraldehyde on the other hand totally suppressed the ability of cells to readjust their volume in hypertonic solutions.By correlating the present data with several earlier published results on CHO cells' posthypertonic survival, it appears that intracellular K+ plays a key role in the chain of reactions that enable nucleated mammalian cells to sustain hypertonic stresses. Whether intracellular K+ is equally important for the survival of cells at subzero temperatures remains to be determined.

ClC-3 and IClswell are Required for Normal Neutrophil Chemotaxis and Shape Change

Polymorphonuclear leukocytes undergo directed movement to sites of infection, a complex process known as chemotaxis. Extension of the polymorphonuclear leukocyte (PMN) leading edge toward a chemoattractant in association with uropod retraction must involve a coordinated increase/decrease in membrane, redistribution of cell volume, or both. Deficits in PMN phagocytosis and trans-endothelial migration, both highly motile PMN functions, suggested that the anion transporters, ClC-3 and ICl(swell), are involved in cell motility and shape change ( Moreland, J. G., Davis, A. P., Bailey, G., Nauseef, W. M., and Lamb, F. S. (2006) J. Biol. Chem. 281, 12277-12288 ). We hypothesized that ClC-3 and ICl(swell) are required for normal PMN chemotaxis through regulation of cell volume and shape change. Using complementary chemotaxis assays, EZ-TAXIScantrade mark and dynamic imaging analysis software, we analyzed the directed cell movement and morphology of PMNs lacking normal anion transporter function. Murine Clcn3(-/-) PMNs and human PMNs treated with anion transporter inhibitors demonstrated impaired chemotaxis in response to formyl peptide. This included decreased cell velocity and failure to undergo normal cycles of elongation and retraction. Impaired chemotaxis was not due to a diminished number of formyl peptide receptors in either murine or human PMNs, as measured by flow cytometry. Murine Clcn3(-/-) and Clcn3(+/+) PMNs demonstrated a similar regulatory volume decrease, indicating that the ICl(swell) response to hypotonic challenge was intact in these cells. We further demonstrated that ICl(swell) is essential for shape change during human PMN chemotaxis. We speculate that ClC-3 and ICl(swell) have unique roles in regulation of PMN chemotaxis; ICl(swell) through direct effects on PMN volume and ClC-3 through regulation of ICl(swell).

A reliable, efficient, and comprehensive approach to assess myocyte remodeling in cardiac hypertrophy and failure

Interest in ventricular remodeling has increased in recent years due to the development of new therapeutic agents which may produce beneficial changes in cardiac anatomy. Data collected with new techniques have provided a clear and consistent picture of myocyte remodeling in cardiac hypertrophy and failure. A maladaptive change in cardiac myocyte shape has been demonstrated in experimental animals with heart failure and in patients with congestive heart failure due to ischemic and idiopathic dilated cardiomyopathy. This article details an approach to collect comprehensive and reliable data on myocyte shape in an efficient manner using isolated myocytes. The methodology employs a Coulter Channelyzer (Model C256, Coulter Electronics, Inc., Hialeah, FL), a light microscope, and an image analysis system. Underlying problems associated with structural assessment of the heart are discussed and effective solutions are outlined.

Measurement of regional myocardial blood flow in rats by unlabeled microspheres and Coulter channelyzer.

A relatively inexpensive, expeditious, new nonradioactive microsphere method for measuring regional myocardial blood flow (RMBF) was developed with unlabeled microspheres and a Coulter Channelyzer. To validate the efficiency of this method, hearts from rats were perfused ex vivo by retrograde aortic cannulation. Unlabeled microspheres of varying size were injected into a side arm in the aortic cannula or added to blood samples collected from the rats. Microspheres were then recovered from the cardiac tissue and blood samples. It was found that > 97% of perfused microspheres (diam > 9.4 microns) were retained in the myocardium and that 94.8 +/- 2.2% of the trapped microspheres were recovered and counted successfully using a Counter Channelyzer. The percent recovery of microspheres from 2- and 0.5-ml blood samples were 95.4 +/- 2.3 and 95.3 +/- 3.1%, respectively. Blood flow to the anterior and posterior halves of the ventricular free walls and septum were measured in six rats; excellent agreements were found between the results yielded by 10-, 15-, and 20-microns unlabeled microspheres injected simultaneously. The transmural flow gradients in the left ventricular free wall estimated by 10- and 15-microns spheres did not significantly differ from each other. Thus the method developed here provides a new alternative for measurement of RMBF, which currently allows at least three measurements for nontransmural gradient RMBF and at least two measurements for transmural gradient RMBF.

Nuclear DNA content and nucleation patterns in rat cardiac myocytes from different models of cardiac hypertrophy

Nuclear DNA content and number of nuclei were examined in cardiac myocytes isolated from controls and rats with volume and pressure overload hypertrophy to determine if haemodynamic overload alters these nuclear parameters. The experimental groups were comprised of normotensive (WKY) and Spontaneously Hypertensive rats (SHR). Additionally, Sprague-Dawley rats with aortic constriction (AC), pulmonary stenosis (PS), myocardial infarction (MI), and 5 month arteriovenous fistulas (F) were studied along with appropriate shams for each of these groups. Nuclear DNA content was measured from DAPI-stained nuclei using an image analysis microdensitometry system. Myocyte volume was measured with a Coulter Channelyzer system. Approximately 83% of the left ventricular myocytes from the SHR and WKY groups contained a diploid DNA content with the remainder being tetraploid. The remaining experimental and sham groups, all female Sprague-Dawley rats (SD), were approximately 93% diploid. The nucleation patterns differed slightly between rat strains with the SHR/WKY expressing approximately 85% binucleation, 14% mononucleation and 5% tri- or tetranucleation. All SD groups, control and hypertrophied, showed approximately 89% binucleation, and 10% mononucleation with the remainder being tri- or tetranucleated. In summary: (1) cardiac myocytes from SHR/WKY strains are predominantley diploid but to a lesser degree than myocytes from SD; (2) nuclear number follows the same pattern with SHR/WKY showing a smaller percentage of binucleated myocytes than SD myocytes; (3) neither the duration, severity, or type of overload caused a significant change in the extent of polyploidy in overloaded hearts from SD rats; and (4) the extent of polyploidy in cardiac myocytes from both the right and left ventricles of SHR and WKY animals does not differ statistically.

Regional increase in isolated myocyte volume in chronic myocardial infarction in cats

Healing of myocardial infarction is associated with hypertrophy of a region surrounding the scar. In order to characterize the pattern of regional hypertrophy after healing of small myocardial infarctions, we used a Coulter Channelyzer to measure directly regional cell volume and light microscopy to measure cell length of isolated myocytes. Acute left ventricular myocardial infarctions were surgically created in adult cat hearts. After healing for 10.4 ± 5.0 months, cells were dissociated by collagenase perfusion. Myocardial cells were isolated from three regions of the infarcted ventricle and the same three anatomical regions of un-operated control hearts: (1) remote from the infarct, (2) non-scarred tissues adjacent to the infarct, and (3) from the infarct. The volume of cells from control hearts was correlated significantly with individual body weight resulting in large inter-animal variations, but small intra-animal variations. Inter-animal comparisons were made by normalizing adjacent and infarct regions to percent change from its remote region. Myocyte volumes from hearts with healed infarcts were increased by 31% in the infarct region and by 20% in the adjacent region, relative to the corresponding regions from control hearts ( P<0.05). Cell lengths were not different from control in any region. Calculated cross-sectional areas followed the same pattern as was observed for cell volumes. We conclude that there is a region of hypertrophy surrounding a small, transmural healed myocardial infarction that is characterized by increased myocyte cross sectional area with no change in cell length. This pattern is typical of the concentric hypertrophy observed with pressure overload rather than eccentric hypertrophy observed with volume overload.

Nuclear size and DNA content in rat cardiac myocytes during growth, maturation and aging

Changes in nuclear volume and DNA content were examined in cardiac myocytes isolated from 21-day-old (weanling, W), 3-month-old (adult, A), and 2-year-old (old, O) rats to document normal parameters for nuclear growth and DNA content. Nuclear volume was calculated from direct measurements of isolated myocyte nuclear profiles and DNA content was measured from DAPI-stained nuclei using an image analysis microdensitometry system. Myocyte volume was measured with a Coulter Channelyzer system. Nuclear volume increased 79% from W to A as a result of an increase in nuclear length. Nuclear width was unchanged. Nuclear volume was not changed from A to O. Approximately 98% of the left ventricular myocytes from all three rat groups contained a diploid DNA content with the remainder of nuclei being tetraploid. The degree of polyploidy increased slightly, but significantly, in right ventricular myocytes from O. Due to the substantially greater increase in myocyte volume relative to nuclear volume, nuclear volume percentage decreased from 3.65 ± 0.28 to 1.64 ± 0.13 from W to A but was unchanged from A to O. To summarize: (1) nuclear volume of rat cardiac myocytes increases significantly during normal physiological growth (W to A) but the rate of nuclear growth is less than that of cell volume; (2) the increase in nuclear size from W to A is not due to an increase in DNA content; (3) cardiac myocytes from Sprague-Dawley rats are predominantly diploid; and (4) there is little change in DNA content of cardiac myocytes from rats of this strain during growth, maturation and aging.

The Na(+)-H+ exchanger is stimulated and cell volume increased in lymphocytes from patients with essential hypertension.

A stimulation of the Na(+)-H+ exchanger has been shown in platelets of hypertensive man and lymphocytes of spontaneously hypertensive rats. In the present paper, human mononuclear leukocytes (HML) were investigated in 12 patients with essential hypertension with regard to the activity of the Na(+)-H+ exchanger and HML volume. The swelling of HML in isotonic sodium propionate was determined using a Coulter Channelyzer. Compared with matched normotensives, the cell volume of HML in a physiological buffer was significantly increased in essential hypertension (P less than 0.05). The amiloride-inhibitable rate of cell swelling in isotonic sodium propionate was also increased in HML from hypertensives. Amiloride (400 mumol/l) abolished the difference in cell volume within 1 min. These data show a functional swelling of HML in essential hypertension, probably due to an activation of the Na(+)-H+ exchanger. If also representative of smooth muscle cells, these findings could explain hypertensive vessel wall hypertrophy, in part, as functional cell swelling.

Antitumor activity in association with thermochromic change of platinum pyrimidine greens against murine and human tumor cells

Antitumor activity in association with thermochromic change of platinum pyrimidine greens against murine and human tumor cells Antitumor activity The cytotoxic activity of each Pt green was evaluated in vitro with mouse leukemia (L1210), mouse sarcoma (S-180), human Burkitt lymphoma (Daudi), human epitheloid carcinoma (HeLa) and human histiocytic lymphoma (U937). L1210, Daudi and U937 were maintained with RPMI-1640 medium containing penicillin (100 unit/liter), streptomycin (100 mg/ml), 20 mM HEPES (N-2-hydroxyethylpiperazine-Y-2ethanesulfonic acid), and 10 or 20% (for Daudi) fetal bovine serum (FCS). HeLa and S-180 were in MEM medium containing these same four materials (the FCS content was 16and 5%, respectively) and 1% non-essential amino acids. Each tumor cell (1 x lO’/ml) was cultured in the presence or absence of Pt greens for 48 h at 37 “C in 5% CO,. Then the number and size distribution of the cells were assayed with a Coulter Channelyzer. Mitomycin C (10 pg/ml) was added to the medium instead of Pt green for a positive control. Activity was expressed as KS0 values defined as the concentration at 50% inhibition of cell growth unless otherwise noted. Tomoko Okada, Takehiko Shimura and Hiroaki (Yohmei) Okuno* National Chemical Laboratory for Indusq, Tsukuba, Zbaraki 305 (Japan)

Change in cardiac myocyte size distribution in aortic-constricted neonatal rats.

Regional changes in cardiac myocyte size and population distribution were examined in Sprague Dawley rats receiving an abdominal aortic constriction at five days of age. At specific time intervals post-constriction, hearts were recovered from constricted animals and weight-matched controls and isolated myocytes were obtained from right and left ventricles using retrograde coronary perfusion with collagenase. Cell volume and cardiac myocyte population distribution curves were determined using a Coulter Channelyzer system. Cell length was measured directly using a Bioquant Image Analysis system. Myocyte cross-sectional area was calculated from cell volume/length. By three months of age, heart weight and heart weight-body weight ratio in constricted animals had increased by approximately 115% (p less than 0.001) in females and 85% (p less than 0.001) in males compared to controls. Between 15 and 90 days of age, the growth response, as indicated by increased cell volume, was approximately 4x greater in constricted females and 2.5x greater in constricted males compared to corresponding controls. This increase was manifested by a shift in the mean size of the myocyte population to the right and a substantial widening of the distribution. Most of the enlargement was due to increased cross-sectional area, with only a moderate contribution from increased cell length. Significant increases in size were seen in both left and right ventricles. By three months of age, a significant interaction was apparent between aortic constriction and sex. The capacity for hypertrophy was greater in the smaller myocytes in female rats of similar age compared to males. The final degree of hypertrophy was similar for male and female rats, possibly indicating a critical upper limit in cell size for cardiac myocytes.

Regional changes in myocyte size during the reversal of thyroid-inducedcardiac hypertrophy

Regional changes in cardiac myocyte size resulting from thyroid treatment, and the subsequent reversal of hypertrophy following removal of the thyrotoxic stimulus, were examined. Isolated myocytes were prepared from weight-matched controls and rats treated with desiccated thyroid hormone for 10 weeks. Cells were collected from the right and left ventricles. Cell volume was determined with a Coulter Channelyzer system. Cell length was measured directly using a phase microscope. Myocyte cross-sectional area was calculated from cell volume/length. After 10 weeks thyroid treatment, cell volume was increased in all regions, especially the right ventricle and the epimyocardium of the left ventricle (P less than 0.01). Although cell length was increased in all regions (N.S.), most of the myocyte hypertrophy was due to an increase in cross-sectional area, especially in the right ventricle (P less than 0.01) and epimyocardium of the left ventricle (N.S.). By 2 weeks post-treatment, a substantial regression of myocyte hypertrophy had occurred. However, heart weight was still significantly larger than control (P less than 0.001), due to an incomplete regression of myocyte volume. This study indicates that thyroid hormones stimulate myocyte hypertrophy by increasing both cross-sectional area and cell length. The response is more pronounced in right ventricles than in left ventricles. Within the left ventricle, epimyocardial cells enlarge the most. At 2 weeks post-treatment, cardiac hypertrophy is still present and myocytes have not completely returned to a normal size.

Comparison of Bone Marrow Progenitors Responsive to Granulocyte-Macrophage Colony Stimulating Factor and Macrophage Colony Stimulating Factor-1

The responsiveness of bone marrow progenitors (BMP) from C3H mice to highly purified or recombinant preparations of Macrophage Colony-Stimulating Factor-1 (CSF-1) and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) was compared by counting the number of colonies (greater than or equal to 50 cells) after 10 days in culture with CSF. Cells responsive to CSF-1 or GM-CSF exhibited maximum colony formation over a wide dose range, although GM-CSF supported colony formation at lower concentrations. The response of BMP to optimal concentrations of CSF-1 was greater than or equal to 5 times greater than the response of BMP to GM-CSF. Analysis of the kinetics of colony formation revealed that, at day 5, the number of BMP responsive to GM-CSF or CSF-1 was approximately equal; the number of CSF-1 colonies increased significantly through day 10, while those cultured in GM-CSF did not. The response of BMP to CSF-1 and GM-CSF was also studied in liquid culture; the differences in yield of mature macrophages was consistent with the differences observed in agar culture. Although both cell populations were shown to be 100% mononuclear by day 7, Coulter Channelyzer analysis of these mature macrophages showed marked differences in cell size distribution. By day 7, cells grown in CSF-1 resulted in a homogeneous population of large cells, whereas GM-CSF cultures showed a heterogeneous distribution. Finally, CSF-1-derived cells possessed increased nonspecific and specific phagocytic capabilities when compared to GM-CSF-derived macrophages. These findings indicate that the actions of GM-CSF and CSF-1 upon the bone marrow compartment results in the generation of mature macrophages which differ morphologically and functionally and may account for the heterogeneity in macrophage populations.

Regional differences in cardiac myocyte dimensions and number in Sprague-Dawley rats from different suppliers.

Previous experiments conducted in this laboratory have indicated that adult Sprague-Dawley rats obtained from Holtzman Company (H) have hearts that are significantly larger than those obtained from Charles River (CR). To investigate potential differences in cell size and number, isolated myocytes were prepared by retrograde coronary perfusion with collagenase. Cell volume (V) was measured with a Coulter Channelyzer, cell length (L) was measured directly, and cross-sectional area (CSA) was calculated from V/L. Cell number was calculated using data from isolated cells and whole-sectioned tissue. Although V for left and right ventricular myocytes was similar in CR and H rats, myocytes from CR tended to be shorter in L (P less than 0.001), but larger in CSA (N.S.) than myocytes obtained from H rats. In both H and CR, left ventricular V and CSA were greater (P less than 0.01) in endomyocardium than epimyocardium and middle myocardium. Values for V and CSA of right ventricular myocytes were less (P less than 0.01) than those from left ventricles in both H and CR. Hearts from H had 19% more myocytes than those from CR (P less than 0.05). Values for cellular dimensions from CR generally showed less variability than those from H. We conclude that significant regional differences in myocyte size are present in rats from both H and CR. Hearts from H rats have significantly more myocytes than those from weight-matched CR rats. Therefore, investigators should be aware that differences in heart weight and myocyte number can be found in rats of the same strain, but obtained from different suppliers.

Triploid Grass Carp Production

Abstract Triploid grass carp (Ctenopharyngodon idella) were produced by application of various cold or heat shocks to the eggs at different time intervals after addition of water to the milt-roe mixture and for varying durations. Up to 87% of grass carp produced were triploids. The optimum treatment was exposure of grass carp eggs, 1 min after mixture of milt and roe, to an instantaneous temperature increase of 12°C above ambient (26°C) for a duration of 3.5 min. Juvenile grass carp were graded into size-groups, and blood samples from 50-100 fish/group were analyzed with a Coulter counter and channelyzer to determine ploidy. Culling those size-groups with poor percentages of triploidy resulted in populations of up to 95% triploids being returned to ponds for further grow-out. Final selection of 100% triploid grass carp (which were to be stocked for aquatic vegetation management purposes) required analysis of each fish that was 150-250 mm total length.

Age dependent change in size distribution of blood cells in fetal and young mice.

The mean corpuscular volume (MCV) and the size distribution of circulating blood cells were determined in the fetal mice of C3H/He strain by using a new electric cell size analyser, Coulter Channelyzer C-256. On the 12th day of gestation, the volume of circulating blood cells was distributed between approximately 240-820 fl (mode at 470 fl), and the MCV was 534.9 +/- 30.1 fl, i. e., ten to eleven times that of adult one. On the 14th day, two types of cell population were observed; one with smaller cell volume and another with larger one corresponded to that of the blood cells on day 12. Therefore, two peaks were observed to be at 140 and 501 fl in the size distribution curve. The cell population with large volume observed on day 12-14 had been almost disappeared by the 16th day of gestation, and the small blood cells became dominant. The MCV of blood cells was then decreased with the development of fetus, from 188.2 +/- 19.3 fl on day 16, to 135.1 +/- 7.3 fl on day 18, and 117.5 +/- 7.2 fl on day 20. The size of blood cells continued to decrease gradually after birth, and became adult range by 8 weeks after birth. The MCV values of the blood cells were 120.9 +/- 8.6 fl, 87.5 +/- 6.2 fl, and 48.7 +/- 0.8 fl for the newborns of 1 day and 7 days old, and the adult mice, respectively. White blood cells were not separated from the blood samples in this study. However, the size distribution and MCV presented above were appeared to be related essentially to the red blood cells, since the number of white blood cells are negligible small compared with that of the red cells.

Regional differences in myocyte size in normal rat heart.

Three independent methods were evaluated in an effort to obtain reliable values for myocyte size in adult Sprague-Dawley rats. Cell volume was determined from isolated myocytes by a Coulter Channelyzer system. Cell volume was also determined from the product of length and cross-sectional area of isolated myocytes. Additionally, myocyte cross-sectional area was measured morphometrically from electron micrographs of whole perfusion-fixed tissue. A major goal was to determine if anatomical methods used to measure cell volume produce values comparable to the more expeditious and objective Coulter Channelyzer method. The results of these experiments showed that myocyte dimensions obtained from all three techniques were similar. The second major objective was to use the above-mentioned techniques to evaluate regional differences in myocyte size. Myocyte cross-sectional area and volume were significantly larger in the endomyocardium than in the epimyocardium of the left ventricle. Right ventricle myocytes had significantly smaller volumes and cross-sectional areas than did left ventricle myocytes. There were no regional differences in cell lengths. We conclude that the Coulter Channelyzer system gives values for isolated myocyte volume that are similar to values obtained with histometric techniques; values for isolated myocyte cross-sectional area were representative of values obtained from myocytes in whole-sectioned tissue; significant regional differences in myocyte size are present in adult rat hearts; and regional variations in myocyte size are due to differences in myocyte cross-sectional area rather than cell length.

Red cell population distributions in healthy dogs

Red blood cells population distributions were studied in puppies and in mature beagle dogs using the Coulter Channelyzer C-1000. Weekly measurements over a period of six months indicated that only minor fluctuations in population distribution indices occurred. Normal range studies showed that although mean cell volume was similar in young and older dogs, young dogs showed an approximately 12.5 per cent greater volume range due to an increased presence of both larger and smaller cells. The red cell population distributions conformed to a single log normal distribution. A mild anaemia was induced in mature dogs. Compensation for this led to transient changes from the usual log normal shape of the red cell population distribution.

The osmotic response of human erythrocytes and the membrane cytoskeleton.

The volumes of human erythrocytes suspended in solutions of varying concentrations of sodium chloride and sucrose were measured by a Coulter Channelyzer Model H4 with appropriate corrections. The cells showed greatly restricted volume changes at osmolarities between 200-700 mOsm. At osmolarities outside this limit, on the other hand, the cells showed nonrestricted volume changes following essentially the predictions of an ideal osmometer. This unexpected volume response was not spuriously due to changes in shape or to a changing orientation of the cells as they traversed the aperture. The restricted volume change observed was abolished when the cells had previously been treated with diamide or had been heated for 60 minutes at 50 degrees C, conditions that are known to disturb the spectrin-actin network. The possibility must be considered that the osmotic behavior of human erythrocytes may be nonideal and that this nonideal behavior is primarily due to mechanical restriction provided by the spectrin-actin network of the membrane cytoskeleton.

Cloning Efficiency of Cultured Human Tumor Cell Lines Measured With the Use of a Coulter Particle Counter&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;

A technique is described by which colony formation in agarose may be rapidly and reproducibly determined with the use of a modified Coulter particle counter (CPC). The cloning efficiency of RD human rhabdomyosarcoma cells after exposure to vincristine sulfate or cisplatin has been compared with the CPC method or by conventional visual counting. These techniques give very similar results. In combination with a Coulter Channelyzer, the CPC technique can be used to evaluate drug effects in colony sizes from a median of 20 to greater than 290 cells. The integrity of colonies formed by 3 colon carcinoma cell lines was examined to determine if this method could be applied to other systems.

Rapid separation of human monocytes and lymphocytes by Sephadex G-10

Many of the interesting functions of human lymphocytes in vitro are known or suspected to be dependent on the presence of monocytes. A practical limiting factor in cell interaction experiments is the ability to separate and recover functional monocytes and lymphocytes rapidly from the same blood specimens. Here we describe a simple, rapid and inexpensive solution to this problem. Incubation at 37 °C on Sephadex G-10 columns consistently depleted monocytes to low levels (<0.1%) and gave maximum lymphocyte yields (> 60%) in a short time. Half the esterase-positive monocytes entering the column may be easily recovered from the beads by a 4°C chilling/shaking technique; by using 0.5% lidocaine, an additional 15% can be recovered. Simultaneously, the G-10-nonadherent lymphocytes are being further separated into T and B cells by E-rosetting. These recovered G-10-adherent cells were above 90% esterase positive, and showed a cell size distribution on the Coulter channelyzer distincly larger than nonadherent lymphocytes. To illustrate the use of this protocol we present a monocyte titration experiment which demonstrated a plateau of optimal pokeweed mitogen-stimulated Ig production at 5–10% monocytes.