Flow Cytometry
Flow Cytometry FAQs
In order to provide better customer support and address customers' questions about FCM antibodies, we have compiled and published answers to the frequently-asked questions (FAQs) about FCM antibodies, which will be continuously updated.
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Both sequences of sample preparation can be applied. Red splitting before staining can reduce the number of cells and make the specific binding efficiency of antibody and antigen higher. Staining and then splitting red can reduce the number of centrifuges, can retain the number of cells to a greater extent, and is more suitable for samples with a small number of cells or rare samples. In addition, staining before cracking red may affect the expression of some indicators, such as CD122, CD200, CD56, CD183, IGM, etc. These indicators suggest that first crack red and then add antibodies, the staining effect will be better. Customers can choose the most appropriate method according to their own situation.
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If the target of the study contains FcR (such as FcrRII/III), it is necessary to incubate anti-FcrRII/III antibody flow antibody, then incubate FcR blocking agent, and finally incubate other antibodies.
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Flow assay is recommended with fresh cells. If you are in a special situation, it is only recommended that you first fix the prepared single-cell suspension with 4% paraformaldehyde for 30min, then wash it twice with PBS, and store it in the cell stain buffer (which can be replaced with 1% BSA PBS) at 4°C, and test it as soon as possible after receiving the reagent.
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Subsequent experiments requiring cell culture procedures require the use of sterile consumables, such as intracellular factor testing or flow sorting experiments.
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It is hardly to do so. The concentration of antibidy was well designed to have the best SINR, increasing of amount will result with high beckground signal.
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Usually, it is difficult to do so. It might be able to detect the biomarker in some treatment group, but the concentration will be lower than the detection limit in more groups.
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The differentiation of Th cells will only be effected by the treatment. The stimulation process is only used to make Th cells to produce enough cytokine for detection.
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Both flow cytometry sorting and magnetic sorting can do so. Flow cytometry sorting can provide better purification but may effect the activity of cells; magnetic sorting can have the best activity with relatively lower purification.
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The choose of stimulation regent is based on the type of sample. For example, the differentiation of RAW264.7 will be based on the treatment drug, stimulation operation, cell line source and cell conditon. You may check https://www.elabscience.cn/resource-flow_cytometry-detail-974 as reference.
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All samples must follow the same operation process except antibody in order to avoid the effect of fixation and permeabilization.
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Normal expression: All blood cells except platelet. Non-hematopoietic tissue; CD44 will not be expressed in normal hepatocyte; CD44 is expressed in Biliary epithelium; the exoression of CD44 could be increased when B cells and T cells were activated or differentiated to be memory cells.
Abnormal expression: Multiple myeloma cells, most lymphomas, chronic lymphocytic leukemia, tumor associated mast cells; the increasing of different splicing forms of CD44 (CD44v4、CD44v5、CD44v6、、CD44v7、CD44v7/8、CD44v10、CD44v3) may show the evolution of tumor. -
It is required to incubate CD86 antibody on ice to keep the activity of cells, but it is possible to incubate CD206 in room temperature after fixation.
