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4 protocols using anti pd l1 percp efluor 710

1

Phenotypic Characterization of MSCs

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C57BL/6 (H-2b) mice-derived MSCs were treated without (control) or with 10% LICM for 3 days; then the cells were blocked with 10% rat serum and incubated with anti-CD80 PerCP-eFluor 710 (eBioscience, cat. no. 46080180), anti-CD86 PerCP-eFluor 710 (eBioscience, cat. no. 46086280), anti-PD-L1 PerCP-eFluor 710 (eBioscience, cat. no. 46598280) and anti-Fas APC (eBioscience, cat. no. 17095180) mAbs. Flow cytometry was performed with BD FACSJazz; and data were analyzed with FlowJo software.
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2

Multiparameter Flow Cytometry Analysis

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For phenotypic analysis, single cell suspensions were stained as previously described 36. For ex vivo Treg cell analysis, anti‐CD25‐PECy5, anti‐Helios‐FITC, anti‐CD8‐PE (from Biolegend, San Diego, CA, USA), anti‐CD4‐APC‐AF750 (from Thermo Fisher Scientific), and anti‐FoxP3‐APC (from eBiosciences, San Diego, CA, USA) were used. For in vitro induced Treg analysis, Zombie Aqua fixable dye, anti‐CD4‐APC and anti‐CD25‐PECy5 from Biolegend were used. For ex vivo DC analysis, cells were blocked with purified anti‐CD16/32, followed by staining with Zombie Aqua, anti‐I‐A/I‐E‐AF488, anti‐CD11c‐AF700, anti‐CD80‐PECy5 (from Biolegend), anti‐CD3‐PE, anti‐TER119‐PE, anti‐CD11b‐VF450, anti‐CD86‐APC, anti‐CD8‐PECy7 (from Tonbo Biosciences, San Diego, CA, USA), anti‐CD19‐PE, anti‐CD49b‐PE, streptavidin‐APCCy7 (from BD Biosciences, San Jose, CA, USA), anti‐CD103‐biotin and anti‐PD‐L1‐PerCP‐eFluor710 (from eBiosciences) were used.
For in vivo transfer experiments, anti‐CD45.1‐AF700, anti‐CD4‐FITC, anti‐CD25‐PECy5, streptavidin‐BV605 (from Biolegend), anti‐Vβ5‐biotin (from BD Biosciences), and anti‐FoxP3‐APC (from eBiosciences) were used for staining ovalbumin (OVA)‐specific T cells.
Samples were acquired in an Attune Acoustic Focusing Flow Cytometer (Thermo Fisher Scientific) and analyzed using flowjo 10.0 software (Tree Star Inc., Ashland, OR, USA).
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Assessing pDC Diversification by Flow Cytometry

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For assessments of pDC diversification, cells were stained with Zombie Violet fixable viability dye (Biolegend), BV711 anti-CD123 (Biolegend, clone 6H6), PE anti-CD80 (BD, clone L307.4), and PerCP-efluor 710 anti-PD-L1 (eBioscience, clone MIH1) antibodies. Data were acquired with an LSR Fortessa (BD Biosciences) flow cytometer and analyzed with FlowJo software (Tree Star). Flow cytometry analyses were performed at the flow cytometry core facility of IRSL (Paris, France).
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4

Assessing pDC Diversification by Flow Cytometry

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For assessments of pDC diversification, cells were stained with Zombie Violet fixable viability dye (Biolegend), BV711 anti-CD123 (Biolegend, clone 6H6), PE anti-CD80 (BD, clone L307.4), and PerCP-efluor 710 anti-PD-L1 (eBioscience, clone MIH1) antibodies. Data were acquired with an LSR Fortessa (BD Biosciences) flow cytometer and analyzed with FlowJo software (Tree Star). Flow cytometry analyses were performed at the flow cytometry core facility of IRSL (Paris, France).
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