Flow Cytometry FAQs

FMO combined with isotype control (FMO-ISO) is recommended for indicators with low expression or less obvious clusters, and only one tube is needed. Taking IFN-γ as an example, "samples of the group expected to express more IFN-γ" as the sample of FMO-ISO, adding other flow antibodies and isotype control antibodies of IFN-γ in addition to IFN-γ, this tube sample is called FMO combined isotype pairs. The other experimental steps are the same as the experimental group, such as dead or alive, fixed broken membrane, etc.

This product is a liquid and does not need to be dissolved with a solvent.

Antibodies packaged in μg need to be pre-tested to titrate the antibody. We did not do the corresponding titration, we need to do the antibody titration experiment according to the recommended dosage in the instructions, the known antibody concentration and the situation of our own cell samples to find out the optimal antibody dosage, so we cannot give the specific number of use.
In general, the number of times each antibody is used in one experiment is a single dye tube, and each full dye tube is added once (there is a biological control and experimental group), that is, each antibody needs to be added at least 3 times in one experiment.

The spontaneous fluorescence of dead cells will be very strong, if the dead cells are excluded without dead dye, the cell population along the diagonal distribution may appear in the scatter plot of the analysis results, affecting the experimental results. Therefore, in order to ensure the accuracy of the experimental results, for tissue samples other than the spleen, it is generally recommended to add dead dye to exclude dead cells. If it is a blood sample, if the sample is fresh and only a few cell surface stains are done, dead cells can also be excluded, but this requires pre-testing to make sure that the sample is not too many dead cells or will not interfere with the results.
We can analyze the dead cell population of the existing data to see whether the distribution of these cells in the fluorescent channel and the corresponding proportion are basically consistent with the living cell population. If they are consistent, we can not do dead cell staining; if they are inconsistent, it is recommended to add dead and dead dyes.

Prepare two tubes of cell flow machine, which are blank tubes of control group cells and treatment group cells. When boarding, all fluorescent channels are opened to see which channel has a positive signal, and the fluorescence of the drug is determined by judging the interference channel of the sample.

It is recommended to directly soak the living tissue in 1%BSA PBS or RPMI1640 medium or special tissue preservation solution, which can be preserved at 4℃ for 24h, and it is best to experiment as soon as possible. It is suggested that teachers do pre-experiments in advance to verify it.

The amount of antibody is mainly related to the volume of fluid and the number of cells. If the number of cells is small, the whole system can be incubated in half, which is 50ul cell suspension +2.5ul flow antibody. However, if there are very few target cells in the sample, a reduction in the number of cells may affect the results, resulting in only a small number of signals being detected.

The ACK Lysis Buffer cannot remove platelets. Because the platelet density is small, the platelets after centrifugation are in the supernatant of the sample and can be removed by discarding the supernatant.

Not recommended, because calcium and magnesium ions can inhibit enzyme activity.Although most of the enzyme reagents on the market will add EDTA to chelate calcium and magnesium ions, but it is still recommended that you do not use HBSS containing calcium and magnesium ions. In addition, if the indicators related to calcium ions need to be tested later, it is recommended to use HBSS without calcium and magnesium ions, such as the detection of calcium ion flow. If you want to test the indicators that need to stimulate activation, you should also be careful to use enzymes that do not contain EDTA, such as IFNγ produced by activated T cells (because EDTA chelates calcium ions, and calcium ions are important trace elements to ensure the activation process of T cells).

① Choose heparin anticoagulant tube; (2) Sample preparation: a. Human peripheral blood sample: dilute red blood cell lysate in advance, and use it now; B. BMC samples: The samples and reagents were rewarmed to 18~20℃ before the experiment was carried out; The ambient temperature of the experiment was controlled at 18~20℃. The collected fresh human blood was separated by PBMC within 1 h. When adding blood, avoid dispersing the stratified liquid level; Do not mix or shake the tube after adding. (3) During blood collection, the blood sample is fully shaken in the heparin anticoagulant tube to avoid blood coagulation (the storage time of the heparin anticoagulant tube is generally not more than 12h); ④ Cracking red time does not exceed 5min, it is recommended to do pre-experiment to find out the best time; ⑤ aseptic operation; ⑥ The stimulation and blocking time are suggested to be explored through pre-experiment.

① All sample tubes should be fixed and broken as in the experimental group, such as blank control, single standard group, fmo-iso; ② The fixed breaking time of each group should not be different for too long. If the sample size is large, it is recommended to operate in batches; ③ For tumor tissue samples, due to the small number of cells for detection, it is necessary to consider increasing the amount of antibodies and the number of computer time should be at least 1 million; ④ It is recommended to add 75um micron aperture filter for secondary filtration to improve the purity and content of immune cells; (5) Due to the longer stimulation blocking time, it is necessary to pay attention to aseptic operation before the end of the stimulation blocking time.