OBTAINING ADHERENT CELLS FROM SPLEEN
Karen S. Hathcock Alfred Singer Richard J. Hodes
INTRODUCTION
The functional significance of adherent, phagocyte- enriched cell populations has been demonstrated for a number of different immune responses. In general, these cells are required for the successful generation of immune responses, but are not themselves the responding cell population. While such populations have been isolated from a number of anatomic sites, further enrichment procedures have generally been based upon such properties as glass adherence, phagocytosis, and radiation resistance. The functions performed by such cell populations have included both "accessory" and antigen- presenting functions in humoral and cell-mediated responses
(1 - 14), as well as in T cell proliferation to concanavalin A (Con A) and phytohemagglutinin (PHA) (15 - 17). This chapter describes a technique to obtain a non-T, non-B, radio-resistant, glass-adherent cell population from mouse spleen cells. This splenic adherent cell (SAC) population fulfills both the ac-
METHODS FOR STUDYING
MONONUCLEAR PHAGOCYTES 89
ISBN 0-12-044220-5
cessory cell and antigen-presenting cell requirements of pri- mary and secondary in vitro antibody responses ( 2 - 5 , 11), permitting the study of cell interactions involving both "self"
recognition and Ir_ gene expression. In addition, SAC popula- tions have been employed in the analysis of T cell-mediated responses to alloantigens and "modified self" specificities (12, 13). Thus, SAC populations isolated by the technique presented are appropriate cell populations for the study of adherent or macrophagelike cells participating in a variety of immune response systems.
II. REAGENTS
A. Preparation of Media and Cells
Eagles minimal essential medium (MEM)
Fetal calf serum (FCS) (inactivated at 56°C for 30 min) 3% L-glutamine
Penicillin - streptomycin HEPES buffer solution (1 M)
Ammonium chloride - K buffer (ACK) Osmotic lysing buffer (18)
B. Cell Adherence and Harvesting
100-mm glass petri dishes (autoclaved for sterility) Plastic pipettes
Versene 1:5000 (GIBCO, Grand Island, New York) 50-ml polyethylene tubes (Falcon 2070)
37°C humidified incubator with 5% CO2 C. Further Enrichment of Adherent Cells
T cell-specific rabbit anti-mouse brain antisera (RaMB) (19) or anti-Thy 1.2 serum (Litton Bionetics, Kensing- ton, Maryland) (2)
Complement (lyophilized guinea pig complement, Grand Island Biological, Grand Island, New York)
Irradiation source: X ray or cesium (Isomedix, Inc., Parsippany, New Jersey)
37°C water bath Roller drum
I I I . PROCEDURE
A. Media and Cell Preparation 1. Media Preparation
(a) Prepare a washing buffer of MEM containing 20 itiM HEPES, 2 τοΜ L-glutamine, 100 U/ml penicillin, 100 yg/ml streptomycin, and 5% FCS (MEM-5).
(b) Prepare an incubation buffer of MEM containing 20 τοΜ HEPES, 2 inM L-glutamine, 100 U/ml penicillin, 100 yg/ml strep- tomycin, and 20% FCS (MEM-20).
2. Cell Preparation
(a) Using reagents at 4°C, prepare a single cell suspen- sion of mouse spleen cells and osmotically lyse the erythro- cytes. Maintain reagents at 4°C unless directions specify otherwise.
(b) Resuspend spleen cells in MEM-20 at 1 spleen : 10 ml MEM-20 (approximately 107 cells/ml).
B. Cell Adherence and Harvesting
(1) Add 5 ml of spleen cell suspension to each 100-mm glass petri dish; swirl each plate gently to spread cell sus- pension evenly over bottom surface. Incubate dishes for 1 hr at 37°C in a humidified incubator maintained with a 5% CO2 atmosphere.
(2) At the completion of this first incubation period, remove dishes from the incubator and wash to remove non- adherent or loosely adherent contaminating cells by gently swirling, removing incubation medium and adding an additional 5 ml of MEM-20 (prewarmed to 37°C). Incubate dishes for an additional 1 hr at 37°C.
(3) At the completion of this second incubation period, remove dishes from the incubator and gently swirl to resuspend any nonadherent or loosely adherent contaminating cells.
After discarding the incubation medium, gently add 4 ml of MEM-5 (prewarned at 37°C). Repeat washing procedure twice.
(4) Versene harvest of adherent cells. (a) After re- moving wash medium (MEM-5) from the final wash step, add 5 ml of Versene (prewarmed to 37°C) to each dish and incubate for 15 min at 37°C. (b) Harvest the adherent cells by repeatedly pipetting ten times Versene over the entire plating surface of the petri dish. Collect and pool the Versene - cell suspen- sions in 50-ml conical tubes containing 10 ml MEM-5 on ice.
Centrifuge cell suspensions at 250 g for 10 min in a refriger- ated centrifuge (4°C) and resuspend cells in MEM-5 to count.
C. Further Enrichment Procedures
(1) To remove contaminating T cells, resuspend the ad- herent cells in either RaMB or anti-Thy 1.2 and incubate on ice for 30 min. Wash by centrifugation in MEM-5 and expose to complement for 30 min at 37°C. Wash twice by centrifuga- tion and count, and resuspend at a final cell concentration of 1 x 107 viable cells/ml in MEM-20.
(2) Irradiate cell suspension with 1000R (x ray or cesium source).
(3) Incubate cells overnight (12 - 18 hr) at 37° in plastic or siliconized glass tubes on a roller drum.
(4) Centrifuge cell suspension, resuspend for culture, and maintain on ice until used. Viable cell recovery is ap- proximately 1% of the initial spleen cell population.
IV. CRITICAL COMMENTS
An essential requirement for the function of a non-T, non-B adherent cell population has been demonstrated in a number of immune response systems. This chapter describes a technique that isolates adherent accessory or macrophagelike cells (SAC) from mouse spleen cells on the basis of their radio-resistance and adherence properties. This procedure is easily adapted to sterile culture systems and consistently yields a functional subpopulation of adherent cells which are predominantly (75%) mononuclear phagocytes.
Viable cell recovery of the adherent, radio-resistant, T cell-depleted SAC populations after overnight incubation is approximately 1% of the initial spleen cell population. The SAC population is itself composed of three identifiable cell subpopulations: (a) 50 - 80% latex-phagocytic cells (macro- phages) ; (b) 8 - 15% nonphagocytic slg+ cells (B cells), and
(c) 15 - 25% nonphagocytic slg", RaMB" cells ("null" cells) (20). In addition, it has been demonstrated that MHC deter- minants encoded in the I-A and the I-E/C subregions are ex- pressed on 40 - 50% of the phagocytic SAC population (21).
Extensive characterization of the functional activities of the SAC population has been carried out for in vitro pri- mary and secondary antibody responses to soluble (2 - 5, 11)
and erythrocyte antigens (unpublished data) and for T cell proliferative responses to soluble antigen (12). In each of these systems, responses were abrogated or significantly re- duced by the removal of adherent or phagocytic cells from the cultured spleen population. Furthermore, in each case, these responses were completely restored by the addition of small
numbers of SAC, demonstrating that the SAC population provides a source of functional accessory cells. The accessory cell ac- tivity of SAC populations for both humoral and proliferative responses has been shown to be mediated by the Ig", Ia+ phago- cytic cells in the SAC populations (3,4). It has also been demonstrated that SAC are highly enriched in the ability to stimulate allogeneic T cell responses in MLR (12) , and that they are functional accessory cells in T cell-mediated CML responses to "modified self" determinants (13).
The cell fractionation procedure described in this chapter is consistently successful in isolating the non-T, non-B radio- resistant, adherent cell population present in mouse spleen cells that provides accessory cell function in a number of im- mune response systems. During the standardization of the con-
ditions necessary to obtain maximal viable cell recovery, critical variables and potential modifications were investi- gated. Since it has been demonstrated that cell adherence properties vary substantially with temperature, incubation and reagent temperatures have been carefully controlled. Specifi- cally, to maximize viable cell recovery plate adherence, com- plement incubation and overnight suspension cultures are car- ried out at 37°C, while all other cell treatments are per- formed at 4°C. MEM-20 (20% FCS) can be successfully used throughout this procedure, but this concentration of FCS has only been shown to be important during the plate adherence in- cubations and the overnight suspension cultures. For all other steps in this procedure, MEM-5 (5% FCS) can be used without changing the SAC recovery.
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