Help Center- Weak fluorescence signals or no fluorescence expression
Possible causes 1：Improper storage or operation of antibody
Suggestions 1：Store at 2~8℃ and protect from prolonged exposure to light, avoid freeze/thaw circles.
Possible causes 2：Fluorescence quenching
Suggestions 2：Fluorescent antibody and sample added with fluorescent antibody should avoid to light.
Possible causes 3：High auto-fluorescence
Suggestions 3：Change the fluorescent dyes to avoid dyes that emit light the same with cell auto-fluorescence.
Possible causes 4：Incorrect dyeing time or temperature
Suggestions 4：Adjust the dyeing time and temperature properly.
Possible causes 5：Staining intracellular proteins with wrong way
Suggestions 5：In order to get the best intracellular protein staining, the correct buffer system should be used for cell immobilization and membrane rupture to detect the proteins presented in cytoplasm or nuclear.
Possible causes 6：Secretory proteins
Suggestions 6：In flow cytometry detect secretory proteins such as cytokines, chemokines and growth factors must be retained in cells.
Possible causes 7：Protein is down regulated, internalized, or shear cut from cells
Suggestions 7：Make sure that the stimulating conditions adopted will not affect protein localization.
Possible causes 8：The target protein is low expressed in the sample
Suggestions 8：For the antigen with low expression, the brightest fluorescent dye must be used in dyeing. Sometimes two step staining can improve sensitivity, for example, using biotin-Labeled antibody first, and then using fluorescence to combine with antibody.
Possible causes 9：Antigens damaged by cell separation or frozen storage
Suggestions 9：Enzyme used to collect cells from solid tissues or from cell dishes may destroy surface proteins, so try to prepare cells with none enzymatic reagents to make sure the reagents will not affect antigens.
Possible causes 10：Abnormal performance of laser
Suggestions 10：Use flow cytometer to set up and track (CS&T) microspheres to check laser calibration and function.
Possible causes 11：Incorrect use of filter
Suggestions 11：Check the excitation wavelengths and emission wavelengths of the fluorescent dyes to make sure that the correct laser and filter used to collect the data.
Possible causes 12：Overcompensation of data
Suggestions 12：Using single staining control and Fluorescence Minus One (FMO) control to set compensation each time.
Possible causes 13：Incorrect cell gate
Suggestions 13：Ensuring the correct setting of cell group, use reactive dyes and set up gate for single cell group can significantly reduce false positive.
Possible causes 14：Incorrect data analysis
Suggestions 14：In order to show the rare cells or dyed gloomy cells excellently, dual parameters were used to observe the cells.
- High background of fluorescence
Possible causes 1: High auto-fluorescence
Suggestions 1: Samples with the same stimulus condition but without any staining are used as controls for auto-fluorescence.
Possible causes 2: Antibodies bind to dead cells
Suggestions 2: Use active dyes to exclude dead cells.
Possible causes 3: Cy5 dyes
Suggestions 3: Cy5 and other blue dyes will combine with some cell Fc receptors, such as mononuclear cells and macrophages, please use other fluorescent dyes.
Possible causes 4: High concentration of antibody
Suggestions 4: Make sure the dosage of the antibody is suitable.
Possible causes 5: The dyeing time is too long
Suggestions 5: According to the expression of the cell protein, optimizing the antibody concentration and incubation time.
Possible causes 6: Insufficient washing
Suggestions 6: Increase washing times after dyeing.
Possible causes 7: Insufficient compensation regulation
Suggestions 7: Using single staining control and Fluorescence Minus One (FMO) control to set compensation for the experiment.
- Fluorescence abnormality
Possible causes 1: Incorrect isotype control concentration
Suggestions 1: Use the same concentration of isotype control as detection antibody.
Possible causes 2: The isotype control and detect antibody from different manufacturers
Suggestions 2: Use the isotype control and the detection antibody from the same manufacturer.
Possible causes 3: Cell adhesion or dead cells are included in the analysis
Suggestions 3: Set up cell group gate and use active dyes to exclude adhesion and dead cells.
Possible causes 4: The immobilized and broken membrane fluid may affect the cell characteristics
Suggestions 4: Adjust the FSC/SSC voltage to keep the cells in the visible range.
Possible causes 5: Stimulation conditions change cell characteristics
Suggestions 5: Use identified cells to set up gate. Adjust the FSC/SSC voltage to keep the cells in the visible range.