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.

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.

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.

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).

Since the sorting buffer contains serum, prolonged storage increases the risk of bacterial contamination. Therefore, it is recommended to prepare it fresh and use it immediately, with the storage time not exceeding one week.

The cell yield from magnetic bead sorting (negative selection) varies significantly due to differences in samples. We cannot guarantee a specific value or range, but we can ensure that the purity of cells sorted using our magnetic bead sorting kit is consistently above 90%.

The size of the sorting magnetic beads is 100 nm.

It is not recommended, as the cell viability deteriorates after thawing, with a large number of cells dying, which significantly affects subsequent experiments.

The homogeneity of the magnetic beads is easily affected by the buffer. Washing the beads with sorting buffer helps maintain good homogeneity in that buffer. Experiments have shown that adding unwashed beads directly to cells can affect the purity and yield of the final sorted cells.