"Panning" for lymphocytes: a method for cell selection.

Abstract

We have developed a simple method for the fractionation of T and B lymphocytes. Plastic dishes coated with antibodies specific for mouse Ig selectively bind splenic B lymphocytes. The adherent cells are easily removed by gentle pipetting; both adherent and nonadherent populations retain immunologic function. In a typical experiment, when 3 X 107 splenic Iymphocytes were added to a 100 X 15 mm plastic dish coated with microgram quantities of anti-Ig, 98% of the nonadherent cells were Ig negative and 97% of the adherent cells were Ig positive. The method is sufficiently sensitive to allow detection and separation of cell types comprising as little as 2% of the total population and can be modified to allow the selection of cells by a double-antibody procedure. We believe that the plastic dish method will be generally useful for fractionating cells on the basis of their cell surface antigens. The ability to fractionate lymphocytes on the basis of surface phenotype has been a major technical advance in the study of the functional diversity of these cells. Many of the methods currently used exploit antibody specificity to separate cells of one type from a mixed population. Cell lysis with antibody and complement is useful only for cell elimination. Separation by affinity column chromatography (1) or fluorescence-activated cell sorting (2) offers the advantage of positive selection by allowing the recovery of both enriched and depleted cell populations.We report a simple and inexpensive method for the separation of B T cell specificity of the serum was determined by complement-dependent lysis of thymocytes and specific immunoprecipitation of T25 (Thy 1 antigen) from thymocyte membranes (5). The cytotoxic titer of this serum was very low (95% of thymocytes were lysed at an antibody dilution of 1:5). Rabbit anti-mouse Ig was raised by repeated subcutaneous injection of DEAE-cellulose-purified mouse Ig in complete Freund's adjuvant. The Ig fraction was purified by ammonium sulfate precipitation (4) and affinity chromatography on Sepharose 4B conjugated with purified mouse Ig (6).Goat anti-rabbit IgG was purchased as serum from SeraSource, Inc., and purified by ammonium sulfate precipitation and affinity chromatography on Sepharose 4B conjugated with DEAE-cellulose-purified rabbit IgG.Normal rabbit IgG was prepared by ion exchange chromatography on Whatman DE-52 ion exchange resin (7).F(ab')2 fragments were prepared by pepsin digestion and gel filtration through Sephadex G-150 (4).Antibodies conjugated with rhodamine were prepared by the method of Cebra and Goldstein (7). Purity of all antibody reagents was determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (8) under reducing conditions.All immunoglobulin preparations were centrifuged at 80,000 X g for 1 hr to remove aggregates, sterilized by Millipore filtration, and stored at 4°.Fetal Calf Serum. Fetal calf serum, purchased from Grand Island Biological Co., was heat inactivated at 560 for 2 hr and tested for mitogenic activity on splenic l...

Cell-Surface Glycoprotein of Oligodendrocyte Progenitors Involved in Migration

“…To isolate AN2-positive cells by panning, the method of Wysocki and Sato (1978) as modified by Barres et al (1992) was used. Bacteriological Petri dishes were coated at 4°C overnight with 10 g /ml rabbit anti-rat antibody in 0.05 M Tris/ HC l, pH 9.5.…”

High levels of in vitro igm rheumatoid factor synthesis correlate with hla–dr4 in normal individuals

“…Total T cell populations were depleted of the suppressor: cytotoxic subpopulations defined by the monoclonal antibody OKT8 (Ortho Diagnostics, Raritan, NJ) by negative selection using the panning technique (13). Plastic 35-mm petri dishes (Falcon #3001; Becton Dickinson, Oxnard, CA) were coated with 40 pg/ml affinitypurified goat anti-mouse Ig (the kind gift of Dr. Ellen Vitetta).…”

Identification of Oncofetal Antigen/Immature Laminin Receptor Protein Epitopes That Activate BALB/c Mouse OFA/iLRP-Specific Effector and Regulatory T Cell Clones

“…Their spleens were excised, minced, washed in complete IMDM medium, and the cells were counted. The spleen cells are separated on monoclonal anti-CD2 Ab-coated petri plates into CD2 ϩ and CD2 Ϫ cells using a modification of the panning technique of Wysocki and Sato (44). The negatively selected CD2 Ϫ cells (T cell-depleted) were x-irradiated at 3000 rad and used as APCs.…”