1. Experimental materials:
Detection tube or 96-well microplate primary antibody (FITC label), taking Anti-CD19/FITC antibody as an example
Buffer: PBS (pH 7.4)
Equipment: pipette, centrifuge, ice box or refrigerator, flow cytometer
2. Note:
The labeled antibody was dissolved and homogenized in PBS before use, and it was confirmed that there was no precipitation. Centrifuge quickly for a few seconds to concentrate all the liquid to the bottom of the tube. The labeled antibody should be stored at 4 ° C in the dark to avoid freezing, due to the fixed preservation of the sample or Longer periods of time may affect the binding between cell surface molecules and antibodies, so it is recommended to use fresh samples whenever possible.
3. Operation method :
1. Taking the flow detection step of rat lymphoid tissue cell surface antigen as an example
Sample preparation: Rat lymphoid tissue pieces were removed, quickly immersed in 10 ml of buffer, and ground with a syringe plunger or with two pre-cooled slides.
The suspension was transferred to a 50 ml conical tube and allowed to stand for a while, so that cell clumps and debris settled to the bottom of the tube and filtered through a nylon mesh into a single cell suspension. The mixture was centrifuged at 300-400 g for 4-5 minutes at 4 ° C, and the supernatant was removed.
If the sample is a spleen cell, add a certain amount of RBC lysis to lyse the red blood cells, or separate the lymphocytes with the separation solution. This is not necessary for other samples. The cells were resuspended by adding 50 ml of buffer, and assayed for cell viability using Trypan Blue as needed.
The cell liquid was centrifuged and the supernatant was removed; the cells were resuspended in an appropriate amount of buffer to 2 x 107 cells/ml. Using a directly labeled primary antibody, add CD19/FITC antibody (0.5-1 ug/106 cells) for 5-10 minutes on ice.
Cell fluorescence staining steps:
1. Add 50 ul of diluted primary antibody to each flow tube or plate microwell; add 50 ul of buffer to the blank tube/well or isotype control tube/well. For optimal antibody concentration testing, a range of 2-0.03ug/106 cells is recommended.
2. Add 50 ul of cell suspension (about 106 cells) to each tube/well and mix gently.
3. Protect from light in the ice bath or incubate in a 4 ° C freezer for 20 minutes. (Note: Some antibodies may require longer incubation times. It is recommended that the experiment be pre-tested.) 4. After the incubation is complete, add buffer (2ml per flow test tube and 200ul per microwell) 4°C Centrifuge at 300-400 g for 5 minutes and discard the supernatant.
5. Repeat the washing process 3 times.
6. Resuspend the cells and check with the upflow instrument.
7. Using a direct-labeled primary antibody, resuspend the cells in 500 ul of buffer and test on the machine. If the primary antibody used is a purified or biotinylated antibody, add 50-100 ul of diluted fluorescently labeled secondary antibody or fluorescently labeled avidin (diluted to the appropriate concentration with buffer) per tube/well in an ice bath or Incubate in the 4 ° C freezer for 15-30 minutes in the dark. Wash the cells twice (refer to methods 4 and 5). The cells were then resuspended in 500 ul of buffer and tested on the machine.
8. Analysis of test results: (Note: For multi-color labeling of multiple cell surface antigens, add fluorescent labeled antibody and follow the above steps for incubation and cell washing; try to keep it in the dark and around 4 °C Under temperature)
Second, taking the flow detection step of human peripheral blood cell surface antigen as an example
1. Add 50 ul of fluorescently labeled or biotin-labeled primary antibody (diluted to the appropriate concentration with buffer) in each flow-through assay tube or plate microwell; in blank tubes/wells or isotype control tubes/ Add 50 ul of buffer to the wells.
2. Add 100 ul of whole blood to each tube and mix gently.
3. Incubate for 15-30 minutes in the dark. (Note: Some antibodies with lower binding capacity may require longer incubation times, and it is recommended that the experimenter conduct pre-tests)
4. Add 2 ml of RBC lysis Buffer to each tube (pre-heat recovery to room temperature) and mix well.
5. Incubate at room temperature for 10 minutes in the dark, and do not exceed 15 minutes.
6. Centrifuge at room temperature 300-400g for 5 minutes and discard the supernatant.
7. Wash the cells once with 2 ml of buffer.
8. Resuspend the cells and check with the upflow instrument.
9. If the primary antibody used is a fluorescent direct-labeled antibody, resuspend the cells in 500 ul buffer or 2% paraformaldehyde fixative and test on the machine. If the primary antibody used is a purified or biotinylated antibody, add 50-100 ul of diluted fluorescently labeled secondary antibody or fluorescently labeled avidin (diluted to the appropriate concentration with buffer) per tube/well and shield from light at room temperature. Incubate for 15-30 minutes. Wash the cells 1-2 times (refer to the method in step 7). The cells were then resuspended in 500 ul or 2% paraformaldehyde fixative and tested on board.
10. Analysis of test results. (Note: For multi-color labeling of multiple cell surface antigens, add fluorescent labeled antibody and follow the above steps for incubation and cell washing; try to keep it in the dark)
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