We have been working with canine peripheral blood cells since last year doing phenotypic study. Howerver since we decided to start fractionation of leukocytes (PBMC or total leukocytes) from the peripheral blood for culture purposes, we start to have problems. We have tested all the available protocols published for fraction for canine PBMC . Although all said their procedure yield a good purity and excelent recovery of PBMC, we did not reach the expected results. When we monitore the cells by flow cytometry as well as by microscopy we always have a very degree of contamination. Granulocytes are the major contaminant always over 20%, changing depending on the sample analysed. We have used so many different gradients for diferential centrifugation with no success. Finally we decided to use total leukocytes for the stimulation assay. We have tested direct lysis of peripheral blood with water, hipotonic citrate solution, and ammonium chrloride treatment. All of them reduce the perce ntage of B cells, checked with anti-canine CD21. Finally we have teste dextran treatment to remove the rbc. Unfortunately the destran treatment also lead to low percentage of B cells as identifyed by anti-CD21 antibody. Does anyone have suggestion that could guide us for futere procedures? Many thanks in advance Olindo
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