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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FMO combined with isotype control (FMO-ISO) is recommended for markers with low expression or less obvious clusters, and only one tube is needed. Taking IFN-γ as an example, use 'samples from the group expected to express more IFN-γ' as the sample for FMO-ISO, adding other flow antibodies and the isotype control antibody of IFN-γ in addition to IFN-γ; this tube is called the FMO combined isotype control. The other experimental steps are the same as the experimental group, such as live/dead staining, fixation and permeabilization, etc.
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Antibodies supplied in μg need to be pre-titrated. We have not performed the corresponding titration, so you need to perform the antibody titration experiment according to the recommended dosage in the instructions, the known antibody concentration, and the condition of your own cell samples to find the optimal antibody dosage; therefore we cannot give a specific number of uses.
In general, each antibody is added once per tube in one experiment (there is a biological control and an experimental group), that is, each antibody needs to be added at least 3 times in one experiment. -
Prepare two tubes for flow analysis, which are the blank tube of the control group cells and the treatment group cells. When acquiring, open all fluorescence channels to see which channel shows a positive signal, and determine the fluorescence of the drug by identifying the interfering channel of the sample.
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It is recommended to soak the live tissue directly in 1% BSA in PBS, RPMI 1640 medium, or a special tissue preservation solution, which can be preserved at 4℃ for 24 h; it is best to perform the experiment as soon as possible. It is suggested that you perform pre-experiments in advance to verify this.
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The amount of antibody mainly relates to the volume of the staining system and the number of cells. If you have fewer cells, you can halve the whole system: use 50 µL cell suspension + 2.5 µL flow antibody (i.e., keep the 5 µL per 100 µL ratio). Note: for 2.5×10⁵ cells you should use 2.5 µL of antibody, NOT 1.25 µL — using only 1.25 µL would halve the antibody concentration and may give weak or false-negative signals. However, if target cells are very rare, reducing cell number may weaken the signal.
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Not recommended, because calcium and magnesium ions can inhibit enzyme activity. Although most enzyme reagents on the market add EDTA to chelate calcium and magnesium ions, it is still recommended not to use HBSS containing calcium and magnesium. In addition, if markers related to calcium ions need to be tested later, it is recommended to use calcium- and magnesium-free HBSS, such as for calcium ion flux detection. If you want to test markers that require stimulatory activation, you should also be careful to use enzymes that do not contain EDTA, such as IFN-γ produced by activated T cells (because EDTA chelates calcium ions, and calcium ions are important trace elements for ensuring the activation process of T cells).
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Antibody isotypes/subclasses are categorized based on the heavy chain structure of immunoglobulins (Ig). In mammals:
5 main Ig classes: IgA (α), IgD (δ), IgE (ε), IgG (γ), and IgM (μ).
Human IgG subclasses: IgG1, IgG2, IgG3, IgG4.
Mouse IgG subclasses: IgG1, IgG2a, IgG2b, IgG3.
In flow cytometry:
IgG subclasses (e.g., anti-CD3, CD4, CD8 for T cells; CD19, CD20 for B cells) are commonly used to detect surface antigens. -
In most cases, the markers for naive CD4+ T cells are CD4+CD44−CD62L+ in mice and CD4+CD45RA+CCR7+ in humans. It is recommended to consult relevant literature to confirm the final detection markers.
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Yes, it is possible. However, it is important to note that the recommended antibody dosage may vary between brands, so the corresponding instructions for use should be followed accordingly. Additionally, for isotype controls, it is advisable to select products from the same brand as the primary antibody.
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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.
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No, this is not recommended. When anticoagulated whole blood is centrifuged, the upper layer is plasma, and the lower layer consists of a "blood cell pellet" (containing red blood cells, granulocytes, platelets, and a small number of white blood cells). After removing the plasma, the remaining sample is predominantly composed of red blood cells and granulocytes, with only a minimal proportion of PBMCs. If you attempt to stain the "blood cell pellet after plasma removal" directly, the excessive red blood cells, high background interference, and low PBMC ratio will result in unreliable data and may even clog the flow cytometer.
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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%.
