Separation of lymphocytes by specific adherence to cellular monolayers containing protein A of Staphylococcus aureus.
A technique was developed for the isolation of lymphocytes according to their surface antigenic markers. The method was based on the general reaction of protein A (SpA from S. aureus) with the Fc-part of an IgG antibody. Monolayers of S. aureus or Spa-coated sheep red blood cells (SpA-SRBC) fix antibody-charged cells specifically; non-fixed cells are easily removed by washing. Alternatively, the monolayers can be treated with a cell surface specific antibody prior to addition of non-charged cells. Monolayer-adhered cells were detached by the addition of lysostaphin (bacterial monolayer) or ammonium chloride (SpA-SRBC monolayer). As an example, Ig-bearing cells were isolated from mouse spleen lymphocytes using a specific rabbit anti-mouse Ig serum for charging either the cells or the monolayers. The recovery of Ig-bearing cells was approximately 84%. The purity of the cells was approximately 83% and the viability 89%. Antibody-SpA complexes on the surface of isolated cells were removed either by trypsin treatment or by cultivation. Ig on the cell surface is restored after 16 hr of cultivation.
D000643:Ammonium Chloride; D000818:Animals; D000936:Antigen-Antibody Complex; D000941:Antigens; D001426:Bacterial Proteins; D002448:Cell Adhesion; D002462:Cell Membrane / Q000276:immunology; D002469:Cell Separation; D002470:Cell Survival; D002478:Cells, Cultured; D004912:Erythrocytes / Q000276:immunology; D005455:Fluorescent Antibody Technique; D007106:Immune Sera; D007141:Immunoglobulin Fc Fragments; D007457:Iodine Radioisotopes; D008214:Lymphocytes / Q000276:immunology*; D008248:Lysostaphin; D008264:Macrophages / Q000276:immunology; D051379:Mice; D008809:Mice, Inbred C3H; D010587:Phagocytosis; D011650:Pulmonary Alveoli / Q000166:cytology; D011817:Rabbits / Q000276:immunology; D011947:Receptors, Antigen, B-Cell; D012756:Sheep / Q000276:immunology; D013154:Spleen / Q000166:cytology; D013210:Staphylococcus / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology; D014357:Trypsin
D000936:Antigen-Antibody Complex; D000941:Antigens; D001426:Bacterial Proteins; D007106:Immune Sera; D007141:Immunoglobulin Fc Fragments; D007457:Iodine Radioisotopes; D011947:Receptors, Antigen, B-Cell; D000643:Ammonium Chloride; D014357:Trypsin; D008248:Lysostaphin