细胞膜表面抗原染色步骤（来自Biolegend）

niwanmao

标题：细胞膜表面抗原染色步骤（来自Biolegend）

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.

Lyse Red Cells:
3.  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.
4.  Stop cell lysis by adding 10 ml Cell Staining Buffer to the tube.  Centrifuge for 5 minutes at 350 x g and discard supernatant.
5.  Repeat wash as in step 2.
6.  Count viable cells and resuspend in Cell Staining Buffer at 5-10 x 10⁶ cells/ml and distribute 100 µl/tube of cell suspension (5-10 x 10⁵ cells/tube) into 12 X 75 mm plastic tubes.

Block Fc-Receptors:
7.  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.

Cell-Surface Staining with Antibody:
8.  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.
10.  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.
11. Repeat step 9.
12. Resuspend cell pellet in 0.5 ml of Cell Staining Buffer and add 10 µl (0.25 µg)/million cells of 7-AAD Viability Staining Solution (BioLegend Cat. #420401) to exclude dead cells.  Note, BioLegend does not recommend use of 7-AAD with either PE-Cy5 or PE-Cy7 antibody conjugates.
13.  Incubate on ice for 3-5 minutes in the dark.
14. Analyze with a Flow Cytometer.

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 add a previously determined optimum concentration of fluorochrome conjugated Streptavidin (SAv) reagent (e.g. 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.

Key Reference:
 
Current Protocols in Cytometry (John Wiley & Sons, New York), Unit 6 Phenotypic Analysis.  
Reagent List:
Cell Staining Buffer (BioLegend Cat. #420201)
Red Cell Lysis Buffer (BioLegend Cat. #420301)
7-AAD Viability Staining Solution (BioLegend Cat. #420401)
Fc-Receptor Block for Mouse Cells (anti-CD16/32, BioLegend Cat. #101302)

empire253

Biolegend公司的细胞表面染色温度要求4℃，固定破膜及细胞内因子染色都是室温。
BD公司的要求普遍是4℃。 若果我用Biolegend的抗体+BD固定破膜液，多少度孵育较好呢？
请教倪老师对此有何看法？