Try reference: Maurice R.G. O'Gorman, et. al. A rapid whole blood lysis technique for the diagnosis of moderate or severe leukocyte adhesion deficiency (LAD). Annals of the NYAS, vol. 677;427-430 (1993). We ended up using PMA only (no ionomycin) and added it directly to whole blood at an optimal final concentration of 120ng/ml and then measured the Beta 2 leukocyte integrin expression by gating on grans, monos or lymphs simply by their corresponding light scatter properties. Above this concentration the density of the integrins on the cell surface continued to increase but you begin to get considerable cell loss. We now routinely use 100ng/ml final (easy to make up) in our current clincal assay. If you would like our laboratory procedure, we will fax it to you. Jane Hughes wrote: Dear Flowers I am setting up an assay to screen for the expression of CD18, 11a,11b and 11c on neutrophils; taking mean peak intensity of resting versus stimulated cells. Is it possible that some of you have used Ca ionophore for this purpose and could advise me as to an appropriate concentration fro stimulation ( or advise a reference). Many thanks Jane Hughes Immunology Section Dept Paediatrics Red Cross Children's Hospital Rondebosch, Cape Town, South Africa
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