Isolation and Analysis of Splenic Dendritic Cells

Spleen mononuclear cells were prepared as previously described [69 (link)]. For studies of cDCs, spleens were treated with deoxyribonuclease I (0.5mg/ml; Worthington Biochemical) and collagenase type 4 (1mg/ml; Worthington Biochemical) for 25 minutes at room temperature, in order to ensure maximal recovery of splenic cDCs. Fluorescently conjugated monoclonal antibodies, anti-mouse B220-Alexa Fluor 700 (RA3-6B2), B220-Pacific blue (RA3-6B2), CD19-FiTC (6D5), CD138-BV605 (281–2), IgD-APCCy7 (11-26c.2a), IgM-PECy7 (RMM-1), TCRβ-Alexa Fluor 700 (H57-597), TCRβ-APC/Cy7 (H57-597), CD4-BV605 (RM4-5), IFNγ-BV421 (XMG1.2), ICOS-PE (7E.17G9), Streptavidin-PE/Cy7, CD45.1-FiTC (A20), CD45.2-Alexa Fluor 700 (104), CD11c-APC (N418), MHCII-Pacific blue (M5/114.15.2), CD8α-PE/Cy7 (53–6.7), CD40-PE (1C10), CD80-PE (16-10A1), CD86-PE (GL1), ICOSL-PE (B7-RP1), PDL1-PE (MIH5), PDL2-PE (TY25), CD49d-Biotin (R1-2), CD11a-FiTC (M17/4), Ki-67-PE (16A8) and Zombie Aqua fixable viability dye were purchased from Biolegend (San Diego, CA). Anti-mouse CD95/Fas-BV421 (Jo2), CXCR5-biotin (2G8), and Bcl6-PerCP/Cy5.5 (K112-91) were purchased from BD Biosciences (Franklin Lakes, NJ). Anti-mouse T-bet-APC (eBio4B10), GL-7-APC (GL-7) and PD1-APC/Cy7 (J43) were purchased from eBioscience. Cell surface and intracellular IFNγ, T-bet and Bcl6 staining was performed as previously described [69 (link),70 (link)].

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Sebina I., James K.R., Soon M.S., Fogg L.G., Best S.E., de Labastida Rivera F., Montes de Oca M., Amante F.H., Thomas B.S., Beattie L., Souza-Fonseca-Guimaraes F., Smyth M.J., Hertzog P.J., Hill G.R., Hutloff A., Engwerda C.R, & Haque A. (2016). IFNAR1-Signalling Obstructs ICOS-mediated Humoral Immunity during Non-lethal Blood-Stage Plasmodium Infection. PLoS Pathogens, 12(11), e1005999.

Publication 2016

deoxyribonuclease I collagenase type 4 B220-Pacific blue CD19-FiTC CD138-BV605 TCRβ-Alexa Fluor 700 TCRβ-APC/Cy7 IFNγ-BV421 ICOS-PE Streptavidin-PE/Cy7 MHCII-Pacific blue CD8α-PE/Cy7 Zombie Aqua fixable viability dye

Anti t Bcl6 Biotin Cd138 Cdcs Cell Collagenase Cxcr5 Cy5 5 Deoxyribonuclease i Fitc Icos Ifnγ Intracellular Monoclonal antibodies Mouse Pdl1 Splenic Streptavidin Tcrβ

Corresponding Organization : German Rheumatism Research Centre

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Variable analysis

independent variables

Treatment of spleen cells with deoxyribonuclease I (0.5mg/ml) and collagenase type 4 (1mg/ml) for 25 minutes at room temperature

dependent variables

Recovery of splenic cDCs
Cell surface and intracellular expression of IFNγ, T-bet and Bcl6

control variables

Spleen mononuclear cells prepared as previously described [69]

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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