Isolation and quantification of class 1 MHC gene mutants in mouse T cells by immunoselection with a magnetic cell sorter (MACS).
A method is described for determining the frequency of cells with a mutation in an autosomal gene coding for a membrane protein. Using a monoclonal antibody to H-2Kk surface antigen and magnetic cell separation (MACS) more than 10(4)-fold enrichment of the H-2Kk negative population was achieved, as tested with artificial mixtures containing a known number of antigen-negative cells. After a second magnetic sorting mutant frequencies as low as 10(-6) could be measured. The number of clonogenic mutants was evaluated by limiting dilution cloning and verification of the mutant phenotype by FACScan (flow cytometry) analysis in a representative number of clones. The spontaneous frequency of H-2Kk deficient mutants was 0.80 x 10(-6), and this increased after irradiation with 6 Gy X rays to 3.38 x 10(-6) within the next 8 weeks. About 50 mutant clones were screened for the presence of other class 1 antigens on the cell surface by FACScan analysis. All mutants continued to express other class 1 antigens.
Wixler
V
V
Institut für Medizinische Strahlenkunde, Universität Würzburg, Germany.
Klarmann
B
B
Begemann
U
U
Gahn
G
G
Lorenz
R
R
Hempel
K
K
D000818:Animals; D000911:Antibodies, Monoclonal; D002455:Cell Division; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D004912:Erythrocytes / Q000502:physiology; D005434:Flow Cytometry / Q000379:methods; D005805:Genes, MHC Class I; D006183:H-2 Antigens / Q000502:physiology; D008280:Magnetics; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D010641:Phenotype; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000166:cytology* / Q000502:physiology*
D000911:Antibodies, Monoclonal; D006183:H-2 Antigens; C024755:H-2K(K) antigen