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Cell Surface Immunofluorescence Staining Protocol

2019.4.22

实验概要

A method of identifying  and enumerating specific cell types in a heterogeneous population of  cells by enhancing the specific staining of desired cells, comprising  contacting a sample from the heterogeneous population of cells with a  labeled primary antibody which recognizes and binds to a desired cell  surface antigen and an unlabeled cross-linking agent which recognizes  and binds to the primary antibody is disclosed.

主要试剂

Cell Staining Buffer [ Details:BioLegend Cat. #420201]

Red Cell Lysis Buffer [ Details: BioLegend Cat. #420301]

7-AAD Viability Staining Solution[ Details: BioLegend Cat. #420403]

TruStain FcX™[ Details: anti-CD16/32, BioLegend Cat. #101319]

实验步骤

1.        Harvest Tissue or Cells:

1)         Obtain desired tissue (e.g. spleen, lymph node, thymus, bone marrow)  and prepare a single cell suspension in Cell Staining Buffer (BioLegend  Cat. #420201). If using in vitro stimulated cells, simply resuspend  previously activated cultures in Cell Staining Buffer and proceed to  Step 2.

2)        Add Cell Staining Buffer up to ~15 ml and centrifuge at 350 x g for 5 minutes, discard supernatant.

2.        Lyse Red Cells:

1)         If necessary (e.g. spleen), dilute 10X Red Blood Cell (RBC) Lysis  Buffer (BioLegend Cat. #420301) to 1X working concentration with DI  water and resuspend pellet in 3 ml 1X RBC Lysis Buffer. Incubate on ice  for 5 minutes.

2)         Stop cell lysis by adding 10 ml Cell Staining Buffer to the tube.  Centrifuge for 5 minutes at 350 x g and discard supernatant.

3)        Repeat wash as in step 2.

4)         Count viable cells and resuspend in Cell Staining Buffer at 5-10 x 10  cells/ml and distribute 100 μl/tube cells/tube) into 12 X 75 mm plastic  tubes.

3.        Block Fc-Receptors:

Reagents  that block Fc receptors may be useful for reducing nonspecific  immunofluorescent staining. In the mouse, purified anti-mouse CD16/CD32  antibody specific for Fcγ R III/II (BioLegend Cat. #101302, clone 93)  can be used to block nonspecific staining of antibodies. In this case,  block Fc receptors by pre-incubating cells with 5-10 μg/ml purified  anti-CD16/32 on ice for 10 minutes. In the absence of an  effective/available blocking antibody for human and/or rat Fc receptors,  an alternative approach is to pre-block cells with excess irrelevant  purified Ig from the same species and same isotype as the antibodies  used for immunofluorescent staining.

4.        Cell-Surface Staining with Antibody:

Add  appropriately conjugated fluorescent, biotinylated, or purified primary  antibodies at predetermined optimum concentrations (e.g. anti-CD3-FITC,  anti-CD4-Biotin, and anti-CD8-APC) and incubate on ice for 15-20  minutes in the dark.

9)        Wash 2X with at least 2 ml of Cell Staining Buffer by centrifugation at 350 x g for 5 minutes.

1)    If using a purified primary antibody, resuspend pellet in residual  buffer and add previously determined optimum concentrations of  anti-species immunoglobulin fluorochrome conjugated secondary antibody  (e.g. FITC anti-mouse Ig) and incubate in the dark for 15-20 minutes. If  using a biotinylated primary antibody, resuspend cell pellet in  residual buffer and add previously determined optimum concentrations of  fluorochrome conjugated Streptavidin (SAv) reagent (e.g. SAv-PE,  BioLegend Cat. # 405204) and incubate on ice for 15-20 minutes in the  dark.

2)   Repeat step 9.

3)    Resuspend cell pellet in 0.5 ml of Cell Staining Buffer and add 5 μl  (0.25 μg)/million cells of 7-AAD Viability Staining Solution (BioLegend  Cat. #420403) to exclude dead cells. Note, BioLegend does not recommend  use of 7-AAD with either PE-Cy5 or PE-Cy7 antibody conjugates.

4)   Incubate on ice for 3-5 minutes in the dark.

5)   Analyze with a Flow Cytometer.

5.        Immunofluorescent Staining of Whole Blood:

1)         Add predetermined optimum concentrations of desired fluorochrome  conjugated, biotinylated, or purified primary antibodies to 100 μl of  anti-coagulated whole blood.

2)        Incubate at room temperature for 15-20 minutes in the dark.

3)         Dilute 10X Red Blood Cell (RBC) Lysis Buffer (BioLegend Cat. #420301)  to 1X working concentration with DI water. Warm to room temperature  prior to use. Add 2 ml of 1X RBC lysis solution to whole blood/antibody  mixture. Incubate at room temperature for 10 minutes.

4)        Centrifuge at 350 X g for 5 minutes, discard the supernatant.

5)        Wash 1X with at least 2 ml of Cell Staining Buffer by centrifugation at 350 x g for 5 minutes.

6)         If using a purified primary antibody, resuspend pellet in residual  buffer and add a previously determined optimum concentration of  anti-species immunoglobulin fluorochrome conjugated secondary antibody  (e.g. FITC anti-mouse Ig) and incubate in the dark for 15-20 minutes. If  using a biotinylated primary antibody, resuspend cell pellet in  residual buffer and determined optimum concentration of fluorochrome  conjugated Streptavidin (SAv) PE, BioLegend Cat. # 405204) and incubate  for 15-20 minutes in the dark.

7)        Repeat step 5.

8)        Resuspend cells in 0.5 ml Cell Staining Buffer or 0.5 ml 2% paraformaldehyde-PBS fixation buffer.

9)        Analyze with a Flow Cytometer.


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