This method analyzes whole blood cells compared to analyzing Ficoll-isolated single nucleated cells, thus taking less time, requiring a smaller sample size, and avoiding the loss of Ficoll-isolated cells, especially B cells. Author: J.E. Colligan et al, Translator: Xuitao Cao et al, This experiment is from the "Compendium of Immunology Laboratory Guidelines".
Operation method
Flow cytometry for the detection of human lymphocytes in whole blood. Move basic program Materials (see Appendix 1 for typed V items) Whole blood, usually anticoagulated with E.D.T.A. PBS, with or without 0.1% (m/V) sodium azide, filtered and sterilized Monoclonal antibody (labeled, unlabeled, or biotinylated), optimal working concentration determined by titration Interlabeled secondary antibodies (unlabeled primary antibodies only) F T T C-labeled streptavidin or P E-labeled streptavidin (for biotinylated primary antibodies only) Lysates: e.g., hypotonic balanced salt solution, deionized water, A C K lysate (Appendix 1), or commercial lysates (e.g., FACs-Iyse, Becton-Dickinson; Immuno-lyse, Coulter; Whole BloodLyse and Fix reagent, Gen Trak) 1 % (m /V ) paraformaldehyde, p H 7.4 12m m X 75m m round bottom centrifuge (Falcon) Beckman J-6M freezing centrifuge with JS-4.2 rotor (or replacement centrifuge) 1 . Collect whole blood from the patient. For patients who are being treated with monoclonal antibody or who have developed human anti-mouse antibody (H A M A ) after treatment with monoclonal antibody, add 3 ml of PBS per 100 ul of whole blood. 2 . When whole blood is obtained from a patient with leukocytosis (O 15X 106 leukocytes/ml), it should be diluted with PBS to approximately 8 XIO6 cells/ml. 3 . Add IOOpil whole blood and an appropriate amount of monoclonal antibody (5 to 20 ul) to a 12 m m X 75 m m centrifuge tube. Cells are labeled by one of the following methods: a. Direct method labeling with one or two fluorescently labeled antibodies. b. Indirect labeling with unlabeled primary antibody and fluorescently labeled secondary antibody. c. Indirect labeling with biotinylated primary antibody and F T T C or P E labeled streptavidin. Controls are set up as unlabeled cells, isotype antibody controls and fluorescently labeled unrelated antibody controls (for indirect labeling). 4. Lysate lysed red blood cells (RBCs) (module & 2, Option 1, commercial lysate as described in the instruction manual). Add 3 to 4 ml of PBS containing 0.1 % sodium azide. Add 3 to 4 ml of PBS containing 0.1 % sodium azide. Centrifuge at 300 g for 8 min. Carefully aspirate the supernatant and wash twice. Add 0.1 ml of 1 % polyformaldehyde (p H 7.4). 5 . Fix the cells. Keep out of light at 4°C (< 1 week). Add 0.6 to 1.O m l of sterile PBS before flow-through. 6 . Set the parameters for forward and side angle scattering to understand the size and physical properties of the cells, respectively (Fig. 8.7. Logarithmic coordinates are preferred for side angle scattering (of course, linear coordinates can also be used). 7 . Gateway techniques allow the identification of mononuclear cells (CD14+ ) and all white cells by fluorescently labeled antibodies. For more product details, please visit Aladdin Scientific website.
