About selection of antibodies for flow cytometry, it is suggested to consider following six aspects:

1. Conforming to basic requirements of antibody selection
Specificity of target protein: determine specific surface marker of the cell, or intracellular marker, and be clear of what you want to test.
Species: antibody selecting should be in strict accordance with species of sample to be test, the antibodies of flow cytometry is substantially free of species cross-reactivity.
Application field: it can be used in the experiment of flow cytometry, if the specification clearly demonstrate that it is tested by FC experiment, it's better to have experiment data and amount description.
Clone Number of antibody: Generally it is suggested to select clone number mentioned in reference literature; for a plurality of clone numbers of same antibody, you can select the clone which is labeled by most types of fluorescence.

2. Determine configuration parameter of flow cytometer
Before experiment of flow cytometry, it needs to determine flow cytometry model to be used according to indicator quantity of simultaneous detection, deploys parameters of instrument and consider following aspects:
Lasers: for normal flow cytometry, it has two kinds of lasers: 488 nm and 633 nm separately; while you purchase flow cytometry, some machine is just installed with 488 nm laser for some reason; it should be remembered that machines with same model may not have same installation of lasers.
Filter: i.e., several detection channels, they determine indicator quantities that you can simultaneously detect.
Actual model of an instrument: it helps to confirm detection channels for another time. Same fluorescence in different flow cytometry, detection channels are not the same sometimes.

3. Suggestion for fluorescence selection
Direct labeling or indirect labeling: for antigen expression of high abundance, it is better to choose direct labeling antibody; for antigen expression of low abundance, it is suggested to consider biotin-labeled antibody.
Intensity of fluorescence: fluorescence itself has difference between strong and weak, it needs to select appropriate fluorescence according to condition of antigen expression(strong or weak) and clustering situation. For example, if antigen expression of abundance is weak, or antigen clustering is insignificant, it is recommended to choose strong fluorescence, like PE, APC; on the contrary, for antigen expression of high abundance or obvious clustering, it is recommended to choose common weak fluorescence, like FITC (strength order of fluorescence: PE> APC> PE / Cy5> PerCP / Cy5.5> FITC).
Selection of similar fluorescence: You can try to select new fluorescent, such as Alexa Fluor, eFluor, etc. Because such kind of fluorescence is quite bright, it has good laser stability, it is not sensitive for pH value, and it is water-soluble (e.g., Alexa Fluor488 has same detection channels with FITC, but the former is obviously better than the latter in fluorescence intensity and light sensitivity).

4. Allocation rule of multicolor fluorescence
In multicolor analyzing program, it is restricted by many factors for selection and allocation of fluorescent antibodies, it is still a difficulty.
No conflict for detection channels: it can only choose one kind of fluorescein for each detection channel, fluoresceins among channels can allocate randomly(e.g. for FL1, FITC-labeled antibody is selected, you cannot select Alexa Fluor488 labeled antibody again; while selection of PE or APC-labeled antibodies can be freely combined for other antibodies in the same experiment group).
Fluorescence intensity: spectrum overlapping of various selected fluorescence should be as minimal as possible; otherwise, it will lead to greater compensation. For example, labeling PE/Cy5 and APC simultaneously, it will lead to over-compensation, it needs to replace with PE/Cy5.5 or PerCP/Cy5.5. The most commonly fluorescence allocation in application is: FITC, PE, PerCP, PerCP/Cy5.5, APC.
allocation of multicolor fluorescence: because of rare types of fluorescein of individual antibody, multicolor analysis is often limited, it also need to consider products from a number of antibody companies to make a plan. Therefore, if it needs to analyze more than 5 colors, it is suggested to consult with technical support in SUNGENE BIOTECH.

5. Antibody amount
For a product, it usually provides multiple types for your purchase, you can determine amount of antibody according to actual quantity of samples and recommended dosage in instruction:
Cell number: typically, the sample quantity is 10^6~10^8 cells for one time of flow cytometry, it is recommended to count cell number of each sample, in order to ensure the best labeling of antibody;
Packaging: if antibody counts test as its measurement unit, it needs to calculate the volume of antibody according to cell suspension volume in each test; for antibodies counting ug as a unit of measure, it needs to calculate volume of antibody added according to instruction.

6.Selection for isotype control antibodies
Isotype control (Isotype Control) is used to eliminate background staining due to nonspecifically binding of antibody to cell surface, it is negative control of flow cytometry experiment in real sense, an essential part of flow cytometry experiment.
Source: for isotype control, it should be same immunoglobulins which are derived from same genus(same hypotype, same subchain and same fluorescently label) of primary antibody, it also requires same dose of usage. Be sure to select accurate isotype control, manufacturers will give product number of isotype control antibody in instruction of fluorescent labeled primary antibody usually;
Purified antibody: If they were purified primary antibody plus fluorescent-labeled secondary antibody, it should select same type of isotype control antibody corresponding to primary antibody (If they were purified CD3+PE-labeled secondary antibody + sample in the sample tube, it should be purified isotype control +PE-labeled secondary antibody + sample in the control tube).