Journal: European Journal of Microbiology & Immunology

Article Title: Impact of enzymatic tissue disintegration on the level of surface molecule expression and immune cell function

doi: 10.1556/EuJMI.2.2012.2.3

Figure Lengend Snippet: Antibodies and 2nd step reagents used for flow cytometry

Article Snippet: Unless otherwise stated, experiments were repeated three times, and one representative result out of three independent measurements is shown. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Antibody Clone Fluorochrome Manufacturer Anti-B7H4 clone 9 PE BioLegend Anti-CD3ε 145-2C11 PE eBioscience Anti-CD4 GK1.5 PE eBioscience Anti-CD8a 53-6.7 PE BD Pharmingen Anti-CD11b M1/70 PE eBioscience Anti-CD11c HL3 FITC BD Pharmingen Anti-CD14 Sa2-8 PE eBioscience Anti-CD19 1D3 PE eBioscience Anti-CD25 PC61 PE BD Pharmingen Anti-CD27 LG.3A10 PE BD Pharmingen Anti-CD28 37.51 PE BD Pharmingen Anti-CD34 RAM34 PE eBioscience Anti-CD38 90 PE BD Pharmingen Anti-CD40 Mar-23 Biotin BD Pharmingen Anti-CD45 30-F11 APC BD Pharmingen Anti-CD45R (B220) RA3-6B2 APC eBioscience Anti-CD49b (Pan-NK) DX5 FITC BD Pharmingen Anti-CD54 (ICAM-1) YN1/1-7 Bio Kindly provided by S. Weiss Anti-CD62L MEL-14 APC BD Pharmingen Anti-CD69 H1.2F3 PE BD Pharmingen Anti-CD70 FR70 PE eBioscience Anti-CD80 (B7.1) 16-10A1 Bio eBioscience Anti-CD86 (B7.2) GL1 Bio BD Pharmingen Anti-CD103 2E7 FITC BD Pharmingen Anti-CD134 (OX40) OX-86 PE eBioscience Anti-CD137 (4-1BB) 17B5 PE eBioscience Anti-CD137L (4-1BBL) TKS-1 Bio eBioscience Anti-CD154 (CD40L) MR1 PE BD Pharmingen Anti-CD162 (PSGL-1) 2PH1 PE BD Pharmingen Anti-CD178 (FasL) MLF3 PE BD Pharmingen Anti-CD210 (IL-10R) 1B1.3a PE BD Pharmingen Anti-CD252 (OX40L) RM134L PE BioLegend Anti-CD272 (BTLA) 6F7 PE eBioscience Anti-CD273 (PD-2L) TY25 PE BD Pharmingen Anti-CD274 (PD-1L) MIH5 PE eBioscience Anti-CD275 (ICOSL) HK5.3 PE BioLegend Anti-CD278 (ICOS) 7E.17G9 PE eBioscience Anti-CD279 (PD-1) J43 PE BD Pharmingen Anti-CD282 (TLR4) 6C2 PE eBioscience Anti-CD284 (TLR2) UT41 PE eBioscience Anti-F4/80 BM8 APC eBioscience Anti-GITR DTA-1 PE eBioscience Anti-GITRL YGL 386 PE BioLegend Anti-Ly6G (Gr-1) RB6-8C5 PE-Cy7 eBioscience Anti-MHCI (H-2Kb) AF6-88.5 FITC BD Pharmingen Anti-MHCII M5/114.15.2 APC eBioscience Anti-NK1.1 PK136 PE BD Pharmingen Anti-Vβ8 F23.1 PE BD Pharmingen Streptavidin PE BD Pharmingen Open in a separate window Antibodies and 2nd step reagents used for flow cytometry Proliferation assay Collagenase- or dispase-treated splenocytes (5×10 5 ) from TCR/HA transgenic mice were plated in 96-well, round-bottom plates in a total volume of 200 µl IMDM supplemented with 10% FCS, 1% penicillin/streptomycin and 0.25 mM β-mercaptoethanol.

Techniques:

Journal: Inflammation Research

Article Title: ER stress abrogates the immunosuppressive effect of IL-10 on human macrophages through inhibition of STAT3 activation

doi: 10.1007/s00011-019-01261-9

Figure Lengend Snippet: ER stress inhibits IL-10-induced STAT3 Tyr705 phosphorylation. a Macrophages were analyzed for IL-10 receptor (IL10R) expression after treatment with thapsigargin (TG), tunicamycin (TM), or vehicle control for 2 h using flow cytometry. Light grey histogram indicates background staining. b Cells were measured for expression of STAT3 and phosphorylation of STAT3 at Tyr705 and Ser727 after stimulation with 5 ng/mL IL-10 for 15 min using flow cytometry. Light grey histogram indicates background staining. c Macrophages were treated with TG, TM, or vehicle control for 2 h and analyzed for STAT3 expression. Light grey histogram indicates background staining. d , e Macrophages were pre-treated with TG, TM, or vehicle control and stimulated with 5 ng/mL IL-10 for 15 min. Phosphorylation of STAT3 at Tyr705 was analyzed using flow cytometry. Light grey histogram indicates background staining. Representative example ( d ) and pooled MFI data ( e ) from three different experiments, mean + SEM. f Macrophages were pre-treated with TG, TM or vehicle control and stimulated with 5 ng/mL IL-10 and analyzed for mRNA expression of SOCS3 using qPCR (normalized to GAPDH expression, fold increase compared to unstimulated control). Data ( a – d , f ) are representative examples of three independent experiments using different donors

Article Snippet: Macrophages were stained after inducing ER stress for IL-10R expression using anti-IL10Rα-PE antibody (CD210, REA239, Miltenyi Biotec) in PBS containing 0.5% BSA.

Techniques: Expressing, Flow Cytometry, Staining