Brief Description:
Flow cytometry is a fluorescence-based assay technique that simultaneously measures multiple properties of individual cells suspended in solution, such as cell population count and protein abundance. Flow cytometry uses fluorescently conjugated antibodies to directly detect antigens, each corresponding to a different protein within or on the cell surface, enabling rapid, quantitative, and accurate determination of cell properties and providing superior interpretation of cell population heterogeneity, with the main advantage that it can simultaneously analyze multiple parameters and multiple different cell markers in a cell population.
Figure 1 Schematic diagram of the flow cytometry.
Sample Processing:
Sample preparation for flow cytometry is a critical step, and the quality of sample preparation can have a significant impact on the final test results. Common samples are mostly peripheral blood, bone marrow, cerebrospinal fluid, pleural effusion, ascites, lymph nodes, spleen, liver, etc., different types of samples are best processed within 12h, if they cannot be processed in time, 4 preservation, the storage time varies from sample to sample, and it is best not to exceed 48h.
Figure 2 Blood components.
1. After the sample is collected, it is placed in a test tube containing anticoagulant (generally heparin or EDTA);
2. Add 5ml of lymphocyte separation solution (ABS930) to a 15ml sterile centrifuge tube; （Note:Lymphocyte separator should be returned to room temperature before use, 18-25).
3、hank'S solution (ABS9257) or PBS buffer (ABS962) dilute the anticoagulated whole blood sample in a ratio of 1:1 (if the whole blood sample is viscous, the proportion can be increased appropriately);
4. Carefully along the wall tube, close to the separator layer, very slowly add the fresh diluted 4ml whole blood sample on top of the lymphocyte separator; （Note:Remember not to muddy the lymphocyte separator).
5. Carefully put into the centrifuge (horizontal rotor), 4 centrifuge for 20-30min, the speed is 400g-1000g; （Note:The centrifuge brake should be removed and wait for a natural stop).
6. Take out the tube, remember not to vibrate. It can be seen that the upper layer is pale red transparent plasma, followed by a thin white ring, white sanitized monocyte lymphocytes;
7. Discard the upper layer, suck out the PBMC layer, add PBS, and wash 1-2 times. 300g, 10min, centrifugation to remove the supernatant, and add antibodies for staining.
Fig.3 ABS930 human lymphocyte isolate.
Cerebrospinal fluid, pleural effusion, ascitesEqual bodily fluids:
1. The sample is collected into an anticoagulant tube and stored at room temperature or 4;
2. Centrifuge the sample at 1000-1500rpm for 5min, and discard the supernatant;
3. Add 01% bovine serum albumin PBS, centrifugation at 1000-1500rpm for 5min washing;
4. Add an appropriate amount of PBS to resuspend. If there is a fine precipitate, filter it with a 40um cell sieve (300 mesh, blue) ABS7231 and set aside.
Lymph nodes, spleen, liverand other organizations:
Tissue samples are commonly processed into single-cell suspensions by enzymatic digestion and mechanical disruption. The procedure refers to the preparation of cell culture single cells, noting that 800-1000 rpm low-speed centrifugation can be used for 3 min to remove the effects of cell debris.
Closed (abs9476) or (abs9477).
D16 is a low-adsorbed IgG FC receptor III (FCR III), CD32 is FC receptor II (FCR II), CD16 CD32 is expressed in B cells, monocytes, NK cells, granulocytes, mast cells, dendritic cells (low levels), Kupffer cells, immature thymocytes, and certain activated mature T lymphocytes. False-positive or false-negative results are sometimes exhibited in FCR-expressing cells due to FCRS-mediated Ig FC binding.
To block the FC receptor, add 0. per 106 cells in a 100 L staining volume25 g mouse FC receptor blocker, mix well, incubate on ice for 5-10 min, and then stain with antibodies. There is no need for cell washing between this blocking and immunostaining step.
Fixation (ABS9110), permeabilization (ABS9111).
1. Receiving cells to be tested (flow cytometry experiments, the general cell volume is controlled at 5*106-107 cell ml; The amount of cells per tube for flow cytometry can be 100ul), centrifuge at 1000rpm for 10min, and discard the supernatant;
2. Add 500ul 4 pre-cooled fixative;
3. Incubate at room temperature in the dark for 10min;
Centrifugation at rpm for 10min;
5. Discard the supernatant, add PBS to wash once, and centrifuge at 1000rpm for 10min;
6. Discard the supernatant and add 15 ml permeabilization agent;
7. Incubate at room temperature for 10-15min;
Centrifugation at rpm for 5min;
9. Discard the supernatant and add an appropriate amount (about 100ul).Permeabilization agent(Note that permeabilization agent is added instead of PBS) to resuspend the cells;
10. After staining with detection antibodies, analyze them with flow cytometry.
Flow cytometry FAQs
1. Do all samples need to be fixed before testing?
In special cases, it is very late to fix the permeabilization of the cell membrane index, and the sample can be fixed, 4 put it for detection the next day to maintain the stability of the detection index (if the color of the stained antibody is a conjugated dye (PE-Cy5, PE-Cy7, PerCP-Cy5.)5) It cannot be fixed because the coupled dye will be uncoupled when fixed, which will lead to the degradation of the fluorochrome. In addition, in the research process of protein post-translational modifications, such as phosphorylation, acetylation, methylation, etc., fixatives can fix the sites of post-translational modifications, and can also prevent the target sites from being quickly degraded in living cells.
2. What are the advantages of cell staining buffer (ABS9475) compared with PBS?
Staining buffer is a buffered saline solution that can be used as an antibody and cell dilution step, as well as all wash steps for cell surface staining and flow cytometry, containing fetal bovine serum to reduce non-specific binding of antibodies and fluorescein to cells of interest; It also contains sodium azide, which is a metabolic inhibitor that can inhibit the plaque formation and capping of cell surface antigens.
Spotting and capping: In a fluorescent immunolabeling assay, after mixing two fluorescent antibodies to membrane proteins, the two fluorescent protein antibodies are evenly distributed across the plasma membrane over a period of time due to the fluidity of the membrane proteins. As time continues, the antibodies redistribute on the surface of the cell and accumulate in a certain part of the cell, which is known as plaque. After a period of time, the bivalent antibodies cross-link with each other on the surface of the cell membrane, causing the labeled membrane protein to accumulate at one end of the cell, which is a capping phenomenon.
3. Do I still need to do isotype control after using FC receptor blocker?
Isotype control antibodies act as primary antibodies without a specific target by excluding non-specific binding, including: binding of the antibody to the FC receptor on the target cell; Non-specific binding of antibodies to cellular proteins, carbohydrates, and lipids. However, there are many kinds of cells whose FC receptors can bind to the FC segment (cells expressing DC64, CD32, and CD16 have strong binding ability), and isotype antibodies can bind to FC receptors, but the binding ability and primary antibody are the same, and the non-specific binding of antibodies to cellular proteins, carbohydrates and lipids cannot be solved by FC receptor blockers, and some antibodies are difficult to find isotype antibodies in actual experiments. Moreover, FC receptor blockers are relatively inexpensive and are a better choice.