Methods for phenotyping polarized and locomotor human lymphocytes.
Lymphocytes show heterogeneity both in phenotype and in locomotor activity; methods which permit the simultaneous assessment of both are therefore useful. The activation of locomotion may be dependent on interactions between lymphocytes and accessory cells, making it necessary to study mixed cell populations. We describe here two in vitro procedures which do this. Firstly the lymphocyte polarization assay has been adapted by using the alkaline phosphatase-anti-alkaline phosphatase method (APAAP) after fixation with glutaraldehyde. Secondly, we have allowed lymphocytes to invade collagen gels and then digested the gel with collagenase to recover the locomotor population. This can then be phenotyped by immunofluorescence using a fluorescence-activated cell sorter (FACS). Collagenase did not affect the staining pattern with commonly-used markers such as CD3, -4, -8, -19, -29, -45RO and -45RA.
Newman
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Immunology Department, University of Glasgow (Western Infirmary), UK.
Wilkinson
P C
PC
D000469:Alkaline Phosphatase / Q000276:immunology; D015703:Antigens, CD / Q000032:analysis; D002465:Cell Movement; D006649:Histocompatibility Antigens / Q000032:analysis; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D017493:Leukocyte Common Antigens; D016131:Lymphocyte Subsets; D003012:Microbial Collagenase / Q000494:pharmacology
D015703:Antigens, CD; D006649:Histocompatibility Antigens; D000469:Alkaline Phosphatase; D017493:Leukocyte Common Antigens; D003012:Microbial Collagenase