Cell Surface Targets Staining for Flow Cytometry

2018-08-02Author:adminpraise:2

Introduction:

Cell surface markers can be used to define cell subsets based on developmental stage and analyzed by flow cytometry. These surface markers have different forms and functions. For example, CD4 is a surface marker for T helper cells that can be further differentiated based on expression of other chemokine receptors and cluster of differentiation (CD) markers. Live cells stained with antibodies can be sorted based on unique staining patterns and used for additional experiments.



1. Prepare cells

More detail you can view Sample Preparation for Flow Cytometry.
1)  Collect cells, filtere through a 200-mesh sieve and collect the filtrate.Centrifuge at 300 g for 5 min, and discard the supernatant.
2) Add cell staining buffer(or PBS with 0.1% BSA)  to resuspend the sample.


2. Cell Counting

After counting the suspension with a hemocytometer or other instruments, adjust the cell concentration to about 1 × 107/mL.


3. Set Sample and Control

Groups

Tubes

Controls

Blank

Single staining control

Isotype control

FMO

Biological control

Sample

Experimental sample



4. Block Fc Receptor

Block Fc receptors may reduce nonspecific immunofluorescent staining.

For Mouse cells: purified Anti-Mouse CD16/CD32 antibody specific for FcγR III/II can be used to block nonspecific staining of antibodies. Thus, block Fc receptors by pre-incubating cells with 0.5-1µg Anti-Mouse CD16/CD32 in 100 µL volume for 10 min at room temperature.

For Human and Rat cells: Pre-incubate the cells with excess irrelevant purified Ig from the same species and same isotype as the antibodies used for immunofluorescent staining or serums from the same species as the antibody used.


5.  Cell Surface Staining

1). Add the antibody according to the recommended dosage of the instructions and mix well.

2). Incubate at 4°C for 30 min in the dark.


6.  Detection

1). Add cell staining buffer, centrifuge at 300 g for 5 min, and discard the supernatant.

2). Add 200 μL cell staining buffer to resuspend the sample.

3). Adjust instrument parameters,detection.