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USE OF SEPHADEX COLUMNS TO DEPLETE MONONUCLEAR PHAGOTCYES

Robert X. Mi she11 Barbara B. Mi shell

I. INTRODUCTION

Mononuclear phagocytes can be removed from murine and human lymphoid cell suspensions by passage over columns of Sephadex G-10 (1). This procedure is particularly useful for rapid and gentle processing of large numbers of cells under sterile conditions. Recoveries typically range from 30 to 40%

with murine spleen and lymph node cells (1 - 4). Somewhat higher recoveries have been reported with human peripheral blood leukocytes, which were initially purified on Ficoll - Hypaque (5,6). Depletion of macrophages as judged histolog- ically and by phagocytosis of latex particles ranges from 90 to 99% (5 - 9). Analysis with surface markers and by functional criteria (1,6,7,9,10) indicate that approximately normal proportions of T and B lymphocytes are recovered in the effluent population.

In addition to macrophages, G-10 passage removes most antibody-forming cells from spleens of immune mice (1); and

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226 METHODS FOR STUDYING MONONUCLEAR PHAGOCYTES it has been reported that macrophage granulocyte progenitors

(3), some of the cells which mediate antibody-dependent cytol- ysis (2), and recently activated suppressor T cell blasts (11) are also removed.

The procedure has been employed primarily to evaluate the functions of macrophages in the induction of in vitro immune responses. These include proliferative responses to plant mitogens (9,12,13) and alloantigens (5,9), primary humoral immunity to heterologous red cells (1), T-dependent synthetic polymers (7) and T-independent antigens (4), and primary cell- mediated immunity to major and minor histocompatibility anti-

gens (10). G-10 passage has also been used to study the

contributions of macrophages in mediating the immunoregulatory effects of lipopolysaccharide (8,14) and for evaluating and removing suppressor cells from both immune and normal popul- ations (15 - 17).

II. REAGENTS

A. Preparation of Sephadex G-10

Saline, 0.85% NaCl (w/v)

Sephadex G-10 (Pharmacia Fine Chemicals, Piscataway, NJ Flask, 6 liters

Centrifuge tubes, 50 ml, autoclavable

Nitric acid, concentrated Sulfuric acid, concentrated

Sodium bicarbonate, 1% (w/v) in double-distilled water Hydrochloric acid (concentrated acid diluted 1:100 in double-distilled water)

Glass beads (Microbeads subdivision of Cataphote Division, Ferro Corp., Ferro Corporation, Jackson, Mississippi)

250 - 350 ym, Class IV-A No. 456 Unisphere beads 500 - 710 ym, Class IV-A No. 235.5 Unisphere beads Glass test tubes, 11 x 75mm, with stainless steel closures (No. 2005-00013, Bellco Glass Co., Inc., Vineland, New Jersey)

Drying oven

Balanced salt solution (BSS) (18), with penicillin

(50 U/ml), streptomycin (50 yg/ml) and 5 - 10% heat-inactivated fetal calf serum (56°C, 30 min). Approximately 250 ml/column

Sephadex G-10

Glass beads, 1 tube (approximately 5 gm) of each size

Syringe, disposable, 50 ml

Beaker, 150 ml

Tissue culture dish, 60 mm

Three-way stopcock (No. K-75, Pharmaseal Laboratories Division, American Hospital Supply Corp., Gendale, California)

Pasteur pipettes with rubber bulbs Pipettes: 10 ml, 5 ml, 1 ml

Centrifuge tube, graduated, for collection of effluent Clamp and ring stand

Cell suspension, 1.5 x 10 cells/ml in BSS (4 - 6 x 10 cells/column)

III. PROCEDURES

A. Preparation of Sephadex G-10

(1) Place approximately 2 liters of saline in a 6-liter flask and add approximately 250 gm of Sephadex G-10.

(2) Stir the mixture gently, cover the flask, and allow the Sephadex to swell overnight at 4°C.

(3) Remove the saline from the settled Sephadex with suc- tion and gently resuspend the Sephadex in a volume of saline that is approximately four times the estimated bed volume.

(4) Allow most of the Sephadex to settle; remove the saline and the unsettled fine particles of Sephadex with suction.

Repeat this step three or four times or until the fine parti- cles of Sephadex have been removed.

(5) Add a volume of saline equal to 30 - 50% of the esti- mated bed volume. Adjust the ratio of Sephadex to saline such that 40 - 45 ml of slurry contain 30 - 35 ml of packed Sephadex.

Centrifuge a sample at low speed (just enough to lightly pack the gel) to check the ratio and accordingly add or remove saline.

(6) Distribute the slurry in 40 - 45 ml volumes to 50 ml tubes. Loosely cap the tubes. Autoclave at 110°C for 40 min with slow exhaust. It is best to place the rack of tubes in a large pan in the autoclave to protect it against occasional spillage. When the tubes have cooled sufficiently, tighten the caps and store the sterile Sephadex at room temperature indefinitely.

B. Preparation of Glass Beads

(1) Prepare separately each bead size. In a fume hood, soak the beads in equal volumes of concentrated sulfuric acid and nitric acid for 24 hr. Rinse them under gently running tap water for 8 hrs.

(2) Soak the beads in a large volume of 1% sodium bicar- bonate for 24 hr, followed by kO rinses in double-distilled water.

(3) Soak the beads in diluted hydrochloric acid for 24 hr.

Rinse them in double-distilled water until the pH of the rinse

228 METHODS FOR STUDYING MONONUCLEAR PHAGOCYTES water is above 6 and dry them in a drying oven.

(4) Aliquot approximately 5 gm of beads per tube, top the tubes with stainless steel closures and autoclave them at 121°C for 20 min (fast exhaust).

C. Cell Separation

(1) Clamp the 50-ml syringe to the ring stand. Discard the plunger and cover the top of the syringe column with the sterile lid of a 60-mm culture dish. To the tip of the syringe, attach the stopcock in closed position.

(2) First pour the larger glass beads and then the smaller ones into the syringe. It may be necessary to first loosen the beads. This can be done with a sterile 1-ml plastic pipette or glass stirring rod. Rinse the beads from the sides of the syringe by adding approximately 10 ml of BSS that con- tains fetal calf serum and antibodies (BSS-FCS).

(3) Gently pipette the Sephadex on top of the glass beads (one tube containing 40 - 45 ml of slurry per 50-ml syringe).

After the Sephadex has settled, place a beaker under the column and open the stopcock.

(4) Wash the column with a total of 100 - 150 ml

BSS-FCS. When the last of the BSS-FCS has been added to the column, stir the top of the Sephadex with a pipette to level the top of the column.

(5) When all the fluid has penetrated the column, quickly load the cells onto the Sephadex with a Pasteur pipette, taking care not to disturb the top of the column. Once the cells have penetrated the column, continually add small amounts of BSS-FCS until the band of cells is midway down the column.

At this point, gently add 15 - 20 ml of BSS-FCS. Begin collecting the effluent cells into a graduated centrifuge tube when the cells reach the glass bead layer. Collect only the first 10 - 15 ml for best depletions.

(6) Allow any contaminating Sephadex to settle for 2 - 3 min, transfer the cells to another tube and centrifuge at 200 g for 10 min.

IV. CRITICAL COMMENTS

When judged by functional criteria, the degree of depletion is variable. This can be demonstrated by using 2-mercapto- ethanol as a culture medium supplement that enables partially depleted populations to generate primary humoral immune responses in vitro. With more adequate depletion, 2-mercapto- ethanol does not restore immune competence (1, 1 0 ) . The causes of variability are not understood. We have found that spleen cells from mice maintained under conditions that mini- mize bacterial exposure are more difficult to deplete than those from mice maintained under conventional conditions.

Thus cells that have not been stimulated may more easily pass

through the columns. The likelihood that depleted cells still contain some functional mononuclear phagocytic cells or their precursors should be considered in using the G-10 procedure and in evaluating data obtained with it.

Other investigators have sequentially passed cells through two columns (7) and have conducted depletions at 42°C (11, 15) in order to improve depletion. In our experience, similar modifications have not significantly affected the degree of contamination when judged by the aforementioned criteria.

Other approaches for dealing with the problem of residual contamination are to choose cell sources, such as lymph nodes, which may be easier to deplete (3), to dilute out the effects of the contaminating cells by conducting the assay at low cell concentrations, or to use antigens of limited complexity for immune induction since these apparently require more macro- phages (7) .

Because G-10 passage is generally used to evaluate mono- nuclear phagocytic cells, the resulting biological effects, for example, inability of filtered cells to respond to con- canavalin A, are usually attributed to the loss of these cells.

However, since G-10 passage may remove other biologically important adherent accessory cells, interpretations should take this possibility into account.

We have had little experience in trying to recover the adherent population. A method for this purpose is described by Schwartz et al. (6).

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