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Protocol

Flow Cytometry (FC)

Solutions and Reagents
 
1.    PBS: Phosphate Buffered Saline
2.    Incubation Buffer: PBS with 0.5%BSA
3.    Wash buffer: PBS with 1% BSA(or 1% FBS)
4.    Cell fixation: 2% formaldehyde solution
5.    RBC lysis buffer: Red Blood Cell Lysis Buffer
 
Flow Cytometry(FC) Protocol For Peripheral Blood Sample
 
1.    Aliquot 1 x 106 cells by volume in a assay tube per test from peripheral blood sample which was
       anticoagulated by EDTA.
2.    Add 2–3ml wash buffer to each tube and rinse by centrifugation. Repeat.
3.    Resuspend cells in 100 µl incubation buffer in a 5ml assay tube per test.
4.    Add fluorochrome, or biotinylated-conjugated primary antibody at the appropriate dilution (see the
       antibody datasheet for the appropriate dilution, such as 10 μl/Test) to the assay tubes.
5.    After mixing, incubate for 20 minutes at room temperature away from light.
6.    Add 2ml 1×RBC lysis buffer to the tube, after mixing incubate it for 10 minutes away from light for
       dissolution of red blood cells.
7.    Discard the supernatant by centrifugation and repeat wash by centrifugation in 2–3 ml  wash buffer buffer.
8.    Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.
 
Flow Cytometrys(FC) Protocol For Cells Sample
 
1.   Aliquot 1 x 106 cells from pretreated sample in 100ul by volume in a 5ml assay tube per test.
2.   Add fluorochrome, or biotinylated-conjugated primary antibody at the appropriate dilution (see the
      antibody datasheet for the appropriate dilution, such as 10 μl/Test) to the assay tubes.
3.   Wash by centrifugation in 2–3 ml wash buffer.
4.   Resuspend cells in 0.3-0.5 ml PBS and analyze on flow cytometer.
 
Notices:
Since applications vary, results can be optimized through titrating the appropriate dilution by analyzer.

 

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