完整脾脏的流式细胞分析

Cathy S. Yam; Adeline M. Hajjar

doi:10.21769/BioProtoc.834

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Peer-reviewed

Whole Spleen Flow Cytometry Assay

完整脾脏的流式细胞分析

CY Cathy S. Yam email

AH Adeline M. Hajjar email

发布: 2013年08月05日第3卷第15期 DOI: 10.21769/BioProtoc.834 浏览次数: 35614

评审: Fanglian He

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参见作者原研究论文

The authors used this protocol in:

Cover of PLOS Pathogens, featuring study using the protocol.

Oct 2012

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Abstract

In the Whole Spleen Flow Cytometry Assay, we used splenocytes directly ex vivo for stimulation with a variety of TLR ligands. The splenocytes were stimulated for a total of 4 hours, then stained for intracellular cytokines. We then examined cytokine production via flow cytometry. This allowed us to compare the responses of minimally manipulated primary macrophages/monocytes and conventional dendritic cells.

Materials and Reagents

PerCP/Cy5.5 anti-mouse CD11b (Biolegend, catalog number: 101228 )
PE/Cy7 anti-mouse CD11c (BD Bioscience, catalog number: 558079 )
APC/Cy7 anti-mouse CD19 (BD Bioscience, catalog number: 557655 )
APC anti-mouse CD3e (Biolegend, catalog number: 100312 )
FITC anti-mouse TNFa (Caltag, catalog number: RM9011 )
PE anti-mouse IL-12 (BD Bioscience, catalog number: 554479 )
aCD16/32 FcBlock (BD Bioscience, catalog number: 553142 )
FITC anti-mouse CD8 (BD Bioscience, catalog number: 553031 )
PE anti-mouse CD8 (BD Bioscience, catalog number: 553033 )
PerCP/Cy5.5 anti-mouse CD4 (Biolegend, catalog number: 100539 )
PE/Cy7 anti-mouse CD8 (Biolegend, catalog number: 100722 )
APC anti-mouse CD8 (BD Bioscience, catalog number: 553035 )
Ultrapure O111:B4 EC LPS (Life Technologies, Invitrogen^TM, catalog number: tlrl-pelps )
Synthetic Lipid IVa (Peptides International, catalog number: CLP-24006-S )
ODN1826 (Coley Pharmaceuticals)
Frosted glass slides, nonsterile (Fisherbrand, catalog number: 12-550-343 )
Cytofix/Cytoperm Solution (BD Biosciences, catalog number: 554722 )
10x Perm Wash Buffer (BD Biosciences, catalog number: 554723 )
10% Paraformaldehyde (Electron Microscopy Sciences, catalog number: 15712-S )
RPMI 1640 Medium (with glutamax, HEPES, Phenol red) (Life Technologies, catalog number: 72400-120 )
Fetal bovine serum (FBS) (Heat Inactivated) (Hyclone, catalog number: SH30070.03HI )
Pen/Strep (Life Technologies, catalog number: 15140-122 )
Beta-ME (Sigma-Aldrich, catalog number: M-7522 )
NaCl
KCl
KH₂PO₄
Na₂HPO₄
NaOH
NH₄Cl
NaHCO₃
10x Phosphate buffered saline (PBS) (see Recipes)
1x PBSA/azide (PBSA/az) (see Recipes)
RBC Lysis Buffer (see Recipes)
Culture Medium (see Recipes)
Brefeldin A (Sigma-Aldrich, catalog number: B6542 ) (see Recipes)

Equipment

15 ml conical tubes (BD Biosciences, Falcon^®, catalog number: 352196 )
50 ml conical tubes (BD Biosciences, Falcon^®, catalog number: 352070 )
FACS titertube microtubes (Bio-Rad Laboratories, catalog number: 223-9391 )
70 μM filters (BD Biosciences, Falcon^®, catalog number: 352350 )
Tissue culture (TC) dish (100 x 20 mm) (Corning Incorporated, catalog number: 430167 )
96-well round bottom plates (Corning Incorporated, catalog number: 3799 )
BD FACS Canto I (6 color analyzer) flow cytometer: Equipped with 2 lasers; blue laser 488 nm four color detection and red laser 633 nm 2 color detection. Uses FACSDiva software
Centrifuge (Beckman Coulter, model: Allegra X-15R )