Background

4A9.4 reacts with CD45, a 180-220 kDa leukocyte common antigen (LCA). CD45 antigen is expressed at high levels on all hematopoietic cells including T and B lymphocytes, monocytes, granulocytes, NK cells and dendritic cells, but is not expressed on non-hematopoietic cells. CD45 antibody has also been reported to react weakly with mature blood erythrocytes and platelets. CD45 is a protein tyrosine phosphatase receptor that is critically important for T and B cell antigen receptor-mediated activation.*Target of immunosuppressive antibody treatment.

Experimental Methods

• Take 100μl peripheral blood anticoagulated by EDTA and add to the bottom of 5ml tube
• Add 10μl labeled antibody to the bottom of flow tube mixing with the whole blood, incubate for 20 minutes at room temperature away from light
• Add 2 ml1×RBC lysis buffer, incubate for 10 minutes away from light after mixing, dissolve red blood cells (recommended: RBC lysing Solution 10×,Cat.: FXP001);
• Sample tube is set to 1000 rpm centrifugation for 5 minutes, discard the supernatant;
  5.Add 2 ml PBS wash buffer to resuspend the cells, then1000 rpm centrifugation for 5 minutes, discard the supernatant
• Add 0.5 ml PBS wash buffer to resuspend the cells and detect by flow cytometry (sample should be determined on the day on the machine and can also be added fixation overnight at 4 ℃ then measured)
• [PBS wash buffer: PBS +1% FBS +0.1% NaN3; Cat.: FXP005]
• [Cell fixation: 2% formaldehyde solution]

References: 1.Knapp, W et al., eds. (1989) Leucocyte Typing IV: White Cell Differentiation Antigens, Oxford University Press, New Yor

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