I. Sample Preparation
For details, see flow cytometry sample preparation techniques
1. Collect cells, filter through a 200-mesh sieve, collect the filtrate, centrifuge at 300×g for 5 min, and discard the supernatant.
2. Add appropriate amount of Cell Staining Buffer to the cells, and resuspend the cells by gently blowing with a pipette gun.
I.
Cell counting
After counting the suspension with a hemocyte counting board or other instruments, adjust the cell concentration to approximately 1 × 107/mL.
I.
Setting up the experimental group
According to the experimental design, set up the experimental group and add 100 μL of cell suspension to each tube.
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I
. Closure of Fc Receptors
Closure of the Fc receptor reduces non-specific staining during the staining process.
For mouse samples, purified CD16/CD32 Monoclonal Antibody binds to FcγRIII/II, sealing off non-specific staining and reducing the background fluorescence of negative cells to the level of unlabeled cells. Add 0.5~1 μg of pure anti-mouse CD16/32 monoclonal antibody and incubate for 10 min at room temperature.
For rat samples, blocking can be performed with an excess of purified Ig of the same source and isoform as the fluorescent antibody or serum of the same source, or with a commercial Fc receptor blocker.
For human samples, purified CD16 monoclonal antibody can be used as a blocking FCR blocker. Add 1 μg of pure anti-human CD16 monoclonal antibody and incubate for 10 min at room temperature.
I
. Cell surface antibody incubation
According to the experimental design, in addition to the blank control, add 5 μL of the corresponding antibody to the corresponding single-stained tube and whole-stained tube, mix well, and incubate for 30 min at 4℃ away from light.
I
.
Fixation and membrane breaking
1. Dilute and fix the membrane-breaking solution according to the instructions.
2. Add 1 mL of Cell Staining Buffer to each tube, centrifuge at 300×g for 5 min, and discard the supernatant. 3. Add 100 μL of Cell Staining Buffer and resuspend the cells.
3. Add 100 μL of Cell Staining Buffer and resuspend the cells.
4. Add 1 mL of 1× Fixation Working Solution to each tube, mix gently, and incubate for 30 min at 4℃ away from light.
5. Centrifuge at 600 × g for 5 min and discard the supernatant.
6. Add 2 mL of 1 x Permeabilization Working Solution to each tube and mix gently.
7. Centrifuge at 600 × g for 5 min and discard the supernatant.
8. Repeat steps (6) and (7) once.
I. Antibody incubation
1. Add 100 μL of 1×Permeabilization Working Solution and resuspend the cells.
2. Add 5 μL of antibody to the corresponding single-stained and whole-stained tubes and mix well.
3. Incubate for 30 min at room temperature away from light.
4. Add 1 x Permeabilization Working Solution and resuspend the cells.
5. Centrifuge at 600 × g for 5 min and discard the supernatant.
I. On-board testing
1. Add 200 μL Cell Staining Buffer and resuspend the cells.
2. Adjust the instrument parameters and test on the machine.
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