Helper T lymphocyte 17 (Th17) cells and regulatory T lymphocytes (Tregs) belong to the CD4+ T cell subset and play crucial roles in immune responses. Tregs exert immunosuppressive functions, while Th17 cells promote host defense against extracellular pathogens. Studies have shown that there exists a balance between the pro-inflammatory Th17 cells and the immunosuppressive Treg cells. Disruption of this balance may lead to autoimmune diseases. Therefore, in research related to autoimmune diseases, it is common to assess the levels of Th17 cells and Treg cells, often using flow cytometry methods.

DOI: 10.3390/molecules22010134

For a flow cytometry novice, opting for Th17 cell and Treg cell flow cytometry assay kits directly is a convenient and efficient approach. However, even with assay kits available, practical operation may raise some questions due to a weak foundation in flow cytometry knowledge. Below are some common questions and their corresponding solutions collected regarding the use of Multisciences’s Th17 and Treg assay kits, aiming to assist everyone in conducting Treg/Th17 flow cytometry experiments more effectively.

Sample Tube Design

Q1: For multiple sets of samples tested in batches, should single-stained and blank tubes be included in each batch? Can the fluorescence compensation adjustment performed during the first experiment be applied to subsequent experiments?

A1: If the experiments are conducted in batches within a period of two months, and consistent reagents, sample types, and the same flow cytometer are used throughout, the fluorescence compensation data obtained from the single-stained and blank tubes in the initial experiment can be applied to subsequent experiments. However, to ensure the accuracy of the experimental results, it is recommended to conduct experiments in batches, and if feasible, to perform at least three independent experiments.

Q2: Is the blank tube only containing cell samples without any antibodies added? Is a single staining tube used to stain only one fluorescent labeled antibody? Is the experimental procedure for blank tubes and single dye tubes the same as the sample in the instruction manual?

A2: Yes, a blank tube contains only the cell sample without any antibodies, whereas a single-stained tube is stained with a single fluorophore-conjugated antibody. The experimental procedures for blank tubes and single-stained tubes can follow the same steps outlined in the assay kit manual. However, it’s important to note that no antibodies are added to blank tubes, and only one fluorophore-conjugated antibody is added to single-stained tubes. All other steps remain the same.

Q3:What are the experimental tubes, blank tubes, single staining control tubes, and isotype control tubes in the experiment?

A3:

• Experimental tubes: Tubes processed according to the instructions in the manual, with all antibodies stained.
• Blank tubes: Tubes containing only samples without any antibodies stained.
• Single-stained tubes: Tubes containing samples stained with only one fluorophore-conjugated antibody.
• Control tubes: For Th17 experiments, there is an unstimulated control tube where the sample is prepared without the addition of stimulation inhibitors. The subsequent steps for this control tube are the same as those for the experimental group samples.
• Isotype control tubes: Tubes that require staining with isotype control antibodies, primarily used in Treg experiments. These tubes undergo the same preparation steps as the experimental group until the staining with Foxp3 antibody step. For isotype control tubes, the antibody added at this step is the isotype control antibody for Foxp3 (Mouse IgG1 Isotype Control, PE).

Sample Analysis

Q1: I plan to test another antibody CD8 that is not in the reagent kit, and do it together with the Treg experiment. How is the specific operation done together? Is it added together with CD4 and CD25?

A1:It was added together with CD4 and CD25. CD8 is a surface stained antibody, and CD4 and CD25 are also surface stained antibodies. Surface stained antibodies should be stained together, before intracellular and nuclear antibodies.

Q2:If the samples cannot be tested immediately, is it only necessary to add polyformaldehyde and not to add flow staining buffer? The Treg manual does not state that paraformaldehyde can be added, is it the same with Th17 manual?

A2:If immediate flow cytometry analysis is not possible, instead of resuspending the samples in 500 μL of 1× Flow Cytometry Staining Buffer, resuspend them in 500 μL of paraformaldehyde solution at a concentration of 1-4% and store them at 2-8℃ in the dark for up to 24 hours before detection. The procedures for Treg and Th17 experiments are similar, but it’s recommended to perform the analysis on the same day whenever possible, as experiments conducted on the same day tend to yield better results comparatively.

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Partial References For The Product
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