Journal of Virological Methodr, 36 (1992) 185-I 96 0 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/92/%05.00

185

VIRMET 01286

Short Communication

Simultaneous separation and purification of mononuclear and polymorphonuclear cells from the peripheral blood of cats Thomas E. Toth, Beth Smith and Haywood Pyle Virginia-Maryland Regional College of Veterinary Medicine. Virginia Tech, Blacksburg, VA, U.S.A. (Accepted 3 October

1991)

Summary

Peripheral blood mononuclear cells (PBMC) and polymorphonuclear cells (PMNC) play important roles in immunodeficiency diseases and AIDS-like syndromes in cats caused by feline leukemia virus (FeLV) and presumably also when caused by feline immunodeficiency virus (FIV). For comparative or functional studies it is advantageous or necessary to obtain these cells as separate entities from the same sample of an animal. Therefore, we analyzed the technical parameters of obtaining, separating and purifying these cells simultaneously from several blood samples of several cats. Flow cytometric studies with outbred cats indicated that of various cell separation/purification methods, e.g. from lysed whole blood, by Histopaque 1.077 or 1.119 singledensity, or by 1.077/l. 119 double-density centrifugation, the 1.077/ 1.119 double-density centrifugation of diluted whole blood is the most consistent, practical and effective method of yielding both highly purified PBMC and PMNC as separate entities from the same sample. The interface between plasma and 1.077 contained an average 86% PBMC vs. 14% PMNC, and the interface between 1.077 and 1.119 an average of 2% PBMC vs. 98% PMNC. Lymphocytes separated by this method had an average CD4/CD8 T-cell ratio of 2.0. These data indicate that Histopaque 1.077/ 1.119 double-density gradient allows purification and physical separation of lymphocytes and phagocytes from a blood sample, enabling the investigator to examine both cell types from the same sample simultaneously. Mononuclear cell; Polymorphonuclear cell; Purification; Separation; Blood; Cat Correspondence to: T.E. Toth, Virginia-Maryland Tech, Blacksburg, VA 24061, U.S.A.

Regional College of Veterinary Medicine, Virginia

186

Peripheral blood mononuclear cells (PBMC) (Cockerell et al., 1976; Hebebrand et al., 1977; Pedersen et al., 1987; Rojko et al., 1988; Lafrado et al., 1989; Pedersen et al., 1989; Quackenbush et al., 1989; Tompkins et al., 1989) and polymorphonuclear cells (PMNC) (Lewis et al., 1986; Kiehl et al., 1987; Lafrado et al., 1987; Dezzutti et al., 1990) play important roles in immunodeficiency diseases and AIDS-like syndromes in cats caused by FeLV and presumably when caused by FIV. The various methods used by numerous investigators to obtain PBMC, such as Percoll (Tompkins et al., 1989) Histopaque 1.077 (Quackenbush et al., 1989) and Ficoll-Hypaque (Cockerell et al., 1976) gradients, respectively, or to obtain PMNC, such as Ficoll-Hypaque (Lafrado et al., 1989), Histopaque (Kiehl et al., 1987) or Percoll (Lewis et al., 1986) gradients, respectively, aimed to obtain one or the other cell population with less attention paid to whether they were contaminated with each other. These methods did not yield physically separated pure populations of PBMC or PMNC. Such purified populations would be necessary in studying changes taking place simultaneously in both cell populations in the blood of retrovirusinfected cats. Therefore we analyzed the technical parameters for collection, and simultaneous purification and separation of PBMC and PMNC in the peripheral blood of cats. Cats and sample collection Outbred cats, used by other workers at the College in non-infectious studies, negative for feline leukemia virus antigen and feline immunodeliciency virus antibodies were used. Blood (3-8 ml) was drawn from each cat by venipuncture into EDTA vacutainer tubes and used in the various separation; purification experiments. Flow cytometric analysis Stained and unstained cells, separated and purified by the various methods, were scanned on an Epics V, model 752 (Coulter Electronics, Hialeah, FL, U.S.A.), laser flow cytometer and cell sorter. The three parameters studied per cell were: forward angle light scatter (FALS), 90-degree-angle light scatter (90LS) and green fluorescence (GFL). Laser excitation was normally 300 mV at 488 nm using a 5-W Innova 90 Argon Laser (Coherent Inc., Palo Alto, CA, U.S.A.). The scanned data were analyzed with the multiparameter data acquisition and display system (MDADS) of Coulter Electronics (Hialeah, FL, U.S.A.). Forward angle light scatter and GFL was collected as linear integral, 90LS in log integral. Two parameter histograms at 64 x 64 channels resolution of FALS vs. 90LS and single parameter histograms showing cell number per channel as a function of GFL at a resolution of 256 channels were collected. During data acquisition the PBMC and PMNC populations were gated together, to exclude all other cell populations, and 10 000 cells were counted per sample. To define the percentage of PBMC and PMNC in the total population

187

of cells counted a gate was placed between the PBMC and PMNC populations and the MDADS statistical program was used to calculate the corresponding proportions. Separationlpurzjication from lysed whole blood 45 ml of lysis buffer (8 g ammonium chloride, 840 mg sodium bicarbonate, 3 mg EDTA free acid, per liter, pH 7.4) and 5 ml of blood was placed in a 50-ml Nalgene round bottom tube, mixed well and allowed to sit at room temperature for 10 min. The tube was centrifuged for 10 min at 200 x g. The supernatant was aspirated, and discarded leaving approx. 0.5-1.0 ml of supernatant over the pellet. The pellet was resuspended in 10 ml of 0.1 M phosphate-buffered saline (PBS), pH 7.2, and centrifuged again for 10 min at 200 x g. Washing and centrifugation was repeated two more times. The final pellet was resuspended in the appropriate buffer for further analysis. Single-density separation/pur$cation

with Histopaque I .077 or I .I 19

Blood samples were diluted 1:l with 0.1 M PBS and mixed well by inversion. The twice-diluted blood samples were divided into 2 equal volumes and placed into 2 separate conical centrifuge tubes. If the original blood sample was 3 ml, 15 ml tubes, if more than 3 ml, 50 ml tubes were used. The twice-diluted blood samples were carefully underlayed in one of the tubes with equal volume of Histopaque 1.077 and in the other with equal volume of Histopaque 1.119. The tubes were centrifuged at 400 x g for 30 min at room temperature. After centrifugation, the opaque bands containing cells were carefully aspirated with a Pasteur pipet and transferred to separate conical centrifuge tubes. 10 ml of 0.1 M PBS was added and mixed by inversion. The tubes were centrifuged at 250 x g for 10 min, the supernatant aspirated, discarded and the pellets resuspended in RBC lysis buffer (see above). Lysis, and two cycles of washing in PBS followed as described for the whole blood preparation. Cell pellets were resuspended in 0.5 ml of 3% paraformaldehyde in 0.1 M PBS, pH 7.3, and incubated on ice for 30 min. Two cycles of washing in PBS followed. The final pellet was resuspended in 0.5 ml PBS. Double-density separation/pur$cation

with Histopaque I .077/l .I 19

Blood samples were diluted and placed into conical tubes depending on the volume of the original sample as described above. The twice diluted blood samples were underlayed with equal volumes of Histopaque 1.077 and then with equal volumes of Histopaque 1.119. The tubes were centrifuged at 400 x g for 30 min at room temperature. After centrifugation, two opaque bands at the interfaces between plasma and Histopaque 1.077 (plasma/l .077) and Histopaque 1.077 and 1.119, (1.077/l. 119), respectfully, were separately aspirated with a Pasteur pipet and transferred to separate conical centrifuge

188

tubes. From hereon the samples were handled for flow cytometric analysis as described above for the single-density separation/purification steps. Lymphocyte

phenotyping

Mononuclear cells were acquired by the double-density separation/ purification method from the bands at the 1.077/1.119 interfaces. Appropriate dilution of mouse original monoclonal antibodies (donated by Christopher Ackley and Max D. Cooper, University of Alabama, Birmingham, School of Medicine, U.S.A.), specific for CD4 and CD8 surface markers of feline T lymphocytes were added to lo6 cells/tube. The tubes were incubated at 4°C for 20 min, and centrifuged at 400 x g for 5 min. The supernatant was discarded and the cell pellets were washed twice with 0.1 M PBS (pH 7.5) containing 1% BSA and 0.1% NaNs. Appropriate dilution of FITC-conjugated rabbit antimouse IgG was added to the tubes and incubated at 4°C for 20 min. To generate negative control cell samples, the same dilution of FITC conjugated rabbit anti-mouse IgG was added to lo6 cells of each sample in absence of the primary antibody and incubated at 4°C for 20 min. The tubes were centrifuged and washed as above. The cell pellets were resuspended in 100 pl of wash fluid and were analyzed on the flow cytometer. Proportions of CD4 + and CD8 + PBMC were calculated by subtracting the values of the negative control samples from the values of the samples that had been incubated with both the primary and secondary antibodies. Results Flow cytometric

analysis of PBMC

and PMNC from lysed whole blood

Fig. 1 is a representative two-dimensional histogram of the flow cytometric profile of PBMC and PMNC from lysed whole blood. The ratios between these cell populations in the blood of 5 cats are in Table 1. The samples in general had relatively large RBC contamination in spite of lysis of RBC (Fig. 1) and the TABLE 1 Flow cytometric Cat No.

3174 3175 3177 3185 3191

analysis of mononuclear

and polymorphonuclear

Proportions

cells from lysed whole blood

of cells

Mononuclear

Polymorphonuclear

21 40 15 27 60

79

t

!

;

The two types of cells could not be separated physically. They appeared as distinct cell populations only on flow cytometry histograms.

189

GL

GU

Fig. 1. Two-parameter histogram of cell populations obtained from lysed whole blood.

two cell populations were contaminated considerable degree (Table 1).

with each other to a variable and

Flow cytometric analysis of PBMC and PMNC from single-density (1.077 or

I .I 19) gradients Flow cytometric profile of cells obtained from the 1.077 gradients is represented by Fig. 2A and the proportions of the two cell types in the blood of 5 cats are in Table 2A. Only one band developed in 1.077 gradients, with relatively high degree of RBC contamination (Fig. 2A). In some samples the cells were overwhelmingly PBMC (Table 2A). However, there was absolutely no consistency in this respect. In other samples PBMC were severely contaminated with PMNC and in the case of at least one cat (3189) the ratios were completely reversed (Table 2A). Flow cytometric profile of cells obtained from the 1.119 gradients are TABLE 2A Flow cytometric analysis of mononuclear and polymorphonuclear cells from single-density (1.077) Histopaque gradient Cat No.

Proportions of cells Mononuclear

Polymorphonuclear

3186 3185

z

36 19

3189 3178 3175

761 91

;: 9

Only one band developed; the two types of cells could not be separated physically. They appeared as distinct cell populations only on flow cytometry histograms.

190 TABLE 2B Flow cytometric analysis of mononuclear density (1.119) Histopaque gradient Cat No.

Proportion

and polymorphonuclear

cells from singfe-

of cells in

Upper band

Lower band

Mononuclear

Polymorphonuclear

Mononuclear

Polymorphonuclear

317ja 318@ 3179

0 few 44

few few

11

89

:8

;:

3189 3190

:z

z38

32 62

9:

“Two bands deveioped; efficient separation of the two types of cells could not be achieved because of the close proximity of the bands. During aspiration of the bands, ceils from one contaminated cells from the other.

LI90 Cl

Gl GL

GU

CL

GU

Fig. 2. (A) Two-ureter histogram of cell populations obtained from Hi&opaque 1.077 singi~ensity gradient. (B) Two-parameter histogram of cell populations obtained from Histopaque 1.119 singledensity gradient, upper band. (C) Two-parameter histogram of cell populations obtained from Histopaque 1.I 19 single-density gradient, lower band.

191

represented by Figs. 2B and 2C, and the proportions of the two cell types in the blood of 5 cats are in Table 2B. In contrast to the 1.077 gradients two bands, an upper and a lower one, developed mostly in close proximity. Contamination with RBC was no problem. Upper bands mostly yielded a mixed population of PBMC and PMNC (Fig. 2B) in varying ratios (Table 2B). In case of two cats (3175 and 3186) there were not enough cells in this band for valid analysis of proportions. Lower bands yielded mostly PMNC (Fig. 2C). However, these

-

*.. IC -

GL

GL

:LLs

GU

GU

Fig. 3. (A) Histopaque 1.077/1.119 double-density gradient. Upper band at the plasma/l.077 interface, lower band at the 1.077/l .119 interface. (B) Two-parameter histogram of call populations obtained from Histopaque 1.077/1.119 double-density gradient, upper band. (C) Two-parameter histogram of cell populations obtained from Histopaque 1.077/l .119 double-density gradient, lower band.

192

cells were to a variable, in some instances high (cat 3190) degree contaminated with PBMC (Table 2B). Flow cytometric analysis of PBMC and PMNC from double-density (I.0771 1.119) gradients Two distinct, well separated bands, an upper one at the plasma/l.077 and a lower one at the 1.077/1.119 interfaces, respectively, developed (Fig. 3A). The flow cytometric profile is represented by Figs. 3B and 3C, and the proportion of the two cell types in the blood of 5 cats are in Table 3. The upper band (Figs. 3A and 3B) contained mostly PBMC. Unfortunately, in the case of two cats (3177 and 3 180) whose blood was divided into 6 aliquots to analyze parameters not discussed in this paper, there were not enough cells in this band for a valid analysis (Table 3). The lower bands (Figs. 3A and 3C) showed consistently a very high proportion of PMNC and very low PBMC (Table 3). On the basis of these highly consistent results in the lower bands we assume that those two cats (3177 and 3180) with insufficient numbers of cells in the upper bands would also have had similarly high degrees of separation and proportion of PBMC in the upper bands. Flow cytometric analysis of lymphocyte phenotypes Fig. 4 shows an example of a positive (upper histogram) and negative control (bottom histogram) fluorescence single-parameter histogram. Table 4 shows the proportions and ratios of CD4+ and CD8+ T lymphocytes in the upper bands (plasma/l.077 interface) of the blood samples of 10 cats. These experiments were performed independently after the analysis of the separaTABLE 3 Flow cytometric analysis of mononuclear 1.119) Histopaque gradients Cat No.

3177b 3180b 3182 3174 3181

Proportion

and polymorphonuclear

cells from double-density

of cells in

Upper (plasma/l .077) banda

Lower (1.077/l. 119) banda

Mononuclear

Mononuclear

few (100) 95 92 71

(1.077/

Polymorphonuclear _

Polymorphonuclear 95

J

: 2

ZP

2;

:

z;

“Two distinct, well separated bands (plasma/l .077 and 1.077/l. 119 interfaces) developed. Efficient separation of the two types of cells was achieved by aspirating the bands into separate tubes. bBlood from these two cats was divided into 6 aliquots to analyze 6 parameters (not discussed in this article), resulting in very low numbers of cells per aliquot. As a consequence, very few or no cells were found at the plasma/l.077 interface.

193

LOG INTEGRAL GREEN FLUORESCENCE Fig. 4. Single-parameter

histograms of positive (upper histogram), and negative control (bottom histogram), fluorescence in defining CD4+ and CD8 + PBMC.

TABLE 4 Flow cytometric analysis of CD4+/CD8+ ratios in mononuclear band at the plasma/l.077 interface of histopaque double-density Cat No.

3185 3186 3178 3182 3175 3181 3174 3180 Average

Proportion

cell populations gradients

of cells

CD4 +

CD8+

Ratio

;: 2;

25 17 32

1.8 1.5 1.8

25 13 14 11

;:p’ 2.3 2.6

:Y

:::

37 ;; 41 42

obtained as the

tion/purification methods. No PBMC/PMNC ratio analysis was performed in these tests. Ratios of CD4+/CD8+ T lymphocytes ranged from 1.3 to 2.8 with an average of 2.0. Discussion Although two-dimensional flow cytometry histograms have at times shown both types of cells as distinct populations, whole blood lysis, and single-density Histopaque 1.077 or 1.119 gradients did not allow consistent purification and physical separation of PBMC and PMNC. Single-density Histopaque 1.077

194

gradients developed mostly a single band containing both types, though more PBMC than PMNC, of cells. Single-density Histopaque 1.119 gradients occasionally yielded two bands in close proximity. In some samples these bands could be aspirated separately; in others they were so close to each other that physical separation of PBMC and PMNC was either impossible or very unreliable because during aspiration of the bands cells from one contaminated the other. Double-density Histopaque 1.077/l. 119 gradients consistently yielded two well separated distinct bands that could be harvested efficiently as separate entities. Two-dimensional flow cytometry histograms of the two bands have shown that the upper band, at the plasma/l.077 interface, contained a high proportion of PBMC with little or minimal PMNC contamination, and the lower band, at the 1.077/l. 119 interface, contained an overwhelming proportion of PMNC with minimal PBMC contamination. The ratio of CD4+/CD8 + T-cells (2.0) in the mononuclear cell population obtained from the upper bands of the double-density Histopaque gradients was somewhat higher than the average reported for cats (Pedersen et al., 1989). These data indicate that Histopaque 1.077/l. 119 double-density gradients allow simultaneous purification and physical separation of lymphocytes and phagocytes from the feline blood, enabling the investigator to independently analyze both cell types from the same sample. It is important to note that results of this study were generated with an outbred population of cats. In spite of the natural variation of the PBMC and PMNC of these heterogeneous cats, these cells were consistently purified and separated from each other. As PBMC and PMNC are efficiently separated from each other as well as from the RBC pellet, a high proportion of the total population of both cell types becomes available for experimentation when prepared by our method. References Cockerell, G.L., Krakowka, S., Hoover, R.G., Olsen, R.G. and Yohn, D.S. (1976) Characterization of feline T- and B-lymphocytes and identification of an experimentally induced T-cell neoplasm in the cat. J. Natl. Cancer Res. 57, 907-913. Dezzutti, C.S., Lafrado, L.J., Lewis, M.G. and Olsen R.G. (1990) Inhibition of phorbol esterinduced neutrophil chemiluminescence by FeLV. Arch. Virol. 111, 75-85. Hebebrand, L.C., Mathes, L.E. and Olsen, R.G. (1977) Inhibition of concanavalin A stimulation of feline lymphocytes by inactivated feline leukemia virus. Cancer Res. 37, 45324533. Kiehl, A.R., Fettman, M.J., Quackenbush, S.L. and Hoover, E.A. (1987) Effects of feline leukemia virus infection on neutrophil chemotaxis in vitro. Am. J. Vet. Res. 48, 7680. Lafrado, L.J., Dezzutti, C.S., Lewis, M.G. and Olsen R.G. (1989) Immunodeficiency in latent feline leukemia virus infections. Vet. Immunol. Immunopathol. 21, 39946. Lafrado, L.J., Lewis, M.G., Mathes, L.E. and Olsen, R.G. (1987) Suppression of in vitro neutrophil function by feline leukaemia virus (FeLV) and purified FeLV-pl5E. J. Gen. Virol. 68, 507-513. Lewis, M.G., Duska, G.O., Stiff, M.I., Lafrado, L.J. and Olsen R.G. (1986) Polymorphonuclear leukocyte dysfunction associated with feline leukaemia virus infection. J. Gen. Virol. 67, 21132118. Pedersen, N.C., Ho, E.W., Brown, M.L. and Yamamoto, J.K. (1987) Isolation of T-lymphotropic virus from domestic cats with an immunodeficiency-like syndrome. Science 235, 790-793.

195 Pedersen, NC., Yamamoto, J.K., Ishida, T. and Hansen, H. (1989) Feline immunodeficiency virus infection. Vet. Immunol. Immunopathol. 21, 11 l-129. Quackenbush, S.L., Mullins, J.I. and Hoover, E.A. (1989) Colony forming T lymphocyte deficit in the development of feline retrovirus induced immunodeticiency syndrome. Blood 73, 509-516. Rojko, J., Essex, M. and Trainin, Z. (1988) Feline leukemia/sarcoma viruses and immunodeficiency. Adv. Vet. Sci. Comp. Med. 32, 57-96. Tompkins, M.B., Pang, V.F., Michaely, P.A., Feinmehl, R.I., Basgall, E.J., Bawler, T.V., Zachary, J.F. and Tompkins, W.A.A. (1989) Feline cytotoxic large granular lymphocytes induced by recombinant human IL-2. J. Immunol. 143. 749-752.

Simultaneous separation and purification of mononuclear and polymorphonuclear cells from the peripheral blood of cats.

Peripheral blood mononuclear cells (PBMC) and polymorphonuclear cells (PMNC) play important roles in immunodeficiency diseases and AIDS-like syndromes...
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