Hi Denis this is my current protocol for CMV Ag assay by flow cytometry. If you need more informations feel free to contact me . Edoardo Edoardo Colombo MD Director Flow Lab Dept. of Pathology Valduce Hospital COMO , Italy coledoco@tin.it DETECTION OF CMV Ag ON GRANULOCYTES BY FLOW CYTOMETRY (Edoardo Colombo MD , Carlo Colzani PhD : Valduce Hospital Como , Italy) Note: The Ab reacts with the CMV lower matrix protein pp65 which is the predominant CMV antigen in leukocytes from patients with CMV infection . (DAKO Clone AAC10) ---------------------------------------------------------------------------- -------------- Leukocytes Recovering : 500 無 whole blood are overlaid onto 1 ml Ficoll 1.077 ; let motionless for 20 minutes at room temperature then aspirate the supernatant ( ~ 250 痞) and wash once with PBS 0.01 M ---------------------------------------------------------------------------- -------------- Cells preparation: Fix the cells with 100 無 CALTAG Fix&Perm Kit solution A (or Paraformaldehyde 1%) for 15 minutes at room temperature Wash with PBS 0.01 M , centrifuge and resuspend Permeabilize membranes with 100 無 Fix&Perm Kit solution B (or Triton-X 100 0.1 %) for 15 minutes ; add 10 無 of Anti-CMV Ab (diluted 1:5 - final concentration 0.5 痢/ml) Wash with PBS 0.01 M, centrifuge and resuspend Transfer 100 無 of resuspended cells into CONTROL and TEST tubes ---------------------------------------------------------------------------- ------------------ Staining : Control : Add 10 無 of anti-mouse rabbit FITC antibody for 15 minutes at room temperature in dark Test: Add 10 無 of anti-mouse rabbit FITC antibody for 15 minutes at room temperature in dark Both: After incubation of the secondary antibody the samples are wash and resuspend in 500 無 PBS 0.01 M ---------------------------------------------------------------------------- ------------------- Flow Cytometry Run collecting AT LEAST 50,000 events gated by a FSC/SS bitmap ; if you are testing an immunodepressed patients you may find a high parasitemia , so you can stop the run early , otherwise in not-immunodepressed patients the parasitemia is very low and therefore a large number of events is needed ; if you have RAM trouble ( < 16 MB) don't save tha data as listmode. Remember that anyway the FCS 2.0 standard has problem with file greater than 30 MB (its limit is setted to 99 MB but it doesn't have a CRC for each set of data stored , so you may discover sometimes "crushed" data files...) so a 64 MB RAM is strongly raccomended. The mean of the negative is contained usually in the first decade in my four-decade histogram (I have an Elite ESP) while the positive cells are around channel 7.0 +/- 1.5 ---------------------------------------------------------------------------- ----------------------
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