Flow Cytometry FAQs

CD4 [SK3] and CD4 [RM4-5] are used for cytokine testing. On the one hand, because PMA stimulation causes CD4 endocytosis, CD4 clones SK3 and RM4-5 have less effect on endocytosis. On the other hand, the experimental samples for the detection of cytokines need to be fixed, and the CD4 binding domains of SK3 and RM4-5 are less affected by fixators.

Most mouse macrophage sorting uses F4/80 and CD11b to determine total macrophages, and then uses CD86 to measure M1 type and CD206 to measure M2 type. The above indexes can be used as a reference. If the cells obtained by flow sorting need to be cultured, it is recommended not to use CD206, because CD206 requires a fixed membrane breaking operation, and the cells will all die. In addition, the expression levels of M1 and M2 markers were not high, which was difficult to sort.

It is recommended that the teacher add CD45, CD11b and dead and alive dyes. On the one hand, there are many cell types in tumor samples and the proportion of macrophages is low, making it difficult to find the target group. Dead cells, on the other hand, readily ingest antibodies and probes, leading to apparent nonspecific staining. Moreover, dead cell autofluorescence is particularly strong, which can lead to enhanced background fluorescence, making it impossible to observe weak positive expression of some markers. Therefore, it is suggested that teachers eliminate the interference of dead cells by dead and alive dyes, and then circle CD45 positive to find the target cell population, and finally determine the proportion of macrophages by CD11b and F4/80.

A: The main chemical reagents used are 0.1M PBS, EDTA disodum, NEM, and a reducing agent DTT or 2-MEA, or TCEP, and 10kD ultrafiltration tube or desalination column.
Based on the characteristics of the antibody, it is assumed that the antibody is a conventional hybridoma monoclonal antibody (e.g. mouse IgG 2a,150kDa), and the antibody is reduced in a neutral buffer (e.g. PBS+ EDTA,PH7.2) with appropriate amounts of DTT or 2-MEA, which is exposed to -SH. Then, SMCC activated dye was added to the reduced antibody (according to a certain proportion), mixed at 37℃ or 4℃ overnight, and a chemical kit such as NEM (N-Ethylmaleimide) was added for about 0.5 h to complete the labeling. Determine whether to perform further purification operations based on the actual situation.
In addition, SATA or 2-thiimino-thiane hydrochloride can also be used to treat the antibody, and then add related reagents (SATA requires hydroxylamine hydrochloride, etc.) to activate -SH(sulfhydryl), and then add the SMCC activated dye to the activated antibody (according to a certain proportion), and overnight mixing reaction at 37℃ or 4℃. After adding a chemical kit such as NEM (N-Ethylmaleimide) blocking reaction can also be used, this way will not reduce the antibody, but may interfere with the antigen-antibody binding site, and the steps are more complicated.
The specific choice depends on the type and characteristics of the antibody. The use ratio of fluorescent dyes also needs to be set up for pre-experiment to explore.

The lysate is not recommended for use in poultry. The red blood cells of birds are quite different from those of humans or mice.

Yes, pyrolysis at room temperature (20℃ to 25℃).

The client is advised to use intracellular factor fixation agent, item No. E-CK-A109.

CD16/32 is a specific blocking agent, and the effect is better. Fc receptors are CD16 and D32 proteins on the cell surface, as well as CD64, but CD64 is strongly bound to FcR and can be well bound without specific antibodies.

No, it is recommended to use ultra-pure water, double steaming water, triple steaming water or deionized water dilution. After diluting the film breaker, the cell stain buffer will change the osmotic pressure and salt particle concentration of the sample itself, and eventually lead to cell deterioration or even death.

We have not tested the effect of this ACK on the viability of other cells. At present, in peripheral blood, our main research object is white blood cells. This lysate has little effect on white blood cells under normal conditions.

In this article, we recommend using FMO Combined isotype control as a negative control to set gates. However, FMO Combined isotype control is more complicated, so many experimenters will choose a simpler method and only use isotype control as a negative control to set gates. Isotype controls are used to eliminate non-specific binding of antibodies causing background staining to help find true negative cell populations.