Slam pe cy7

The following antibodies were used for staining: CD4 BUV395, CD8α BUV737 (BD); CD5 PerCp-Cy5.5, CD25 PE or APC, CD44 FITC or Pacific Blue, CD45.2 PE, CD62L APC, CD69 APC, TCRβ Pacific Blue, Vα2 PE or Pacific Blue, CXCR5-biotin, PD-1 FITC, SLAM PE-Cy7, CD95 PE-Cy7, GL-7 FITC (Biolegend); B220 efluore780, CD8α efluore780, ZAP70 Alexa488, Strepavidin-A647 (Life Technology); GP66 tetramer PE or APC (NIH Tetramer Core Facility). The following antibodies were used for immunoblot analysis: ZAP70 (clone 1E7.2) (36 (link)); ZAP70-pTyr³¹⁹ (clone 65E4), LAT-pTyr¹⁹¹, Erk1/2, Erk1/2-pThr²⁰²/pTyr²⁰⁴, and PLCγ-pTyr¹⁸³, all from Cell Signaling Technology; anti-phosphotyrosine (4G10), PLCγ (Sigma-Aldrich), LAT (clone FL-233, Santa Cruz Biotechnology), FLAG (clone M2, Sigma-Aldrich), and GAPDH (clone 6C5, Santa Cruz Biotechnology).

Femora, tibiae and ilia were isolated and gently crushed in staining medium (PBS + 5mM EDTA + 2% fetal bovine serum). Unless stated otherwise, for each untreated (−) sample, the bone marrow of 3 mice were pooled, and for each pI:pC sample the bone marrow of 2 mice were pooled, to increase sample size and to decrease artefacts based on mouse-to-mouse variability. Red blood cells were lysed using Ammonium-Chloride-Potassium (ACK) lysing buffer for 1min on ice. CD117 (c-KIT) positive cells were enriched using CD117 MicroBeads (Miltenyi Biotec) and subsequently immunostained using the antibodies listed here below. Propidium Iodide (ThermoFisher Scientific) was used for negative selection of dead cells. All sorting experiments were performed using BD FACS Aria II or III flow cytometer (BD Biosciences), using the gating strategy illustrated in Figure S1A. Antibodies used for FACS sorting of haematopoietic bone marrow cells: CD3 (Biolegend), CD4 (Biolegend), CD5 (Biolegend), CD8 (Biolegend), Gr1 (Biolegend), CD11b (Biolegend), Ter119 (Biolegend, B220 (Biolegend), anti-rat PeCy5 (Invitrogen), c-kit APC-Cy7 (Biolegend), Sca1 Pacific Blue (Biolegend), SLAM PeCy7 (Biolegend), Flk2 Biotin (Biolegend), CD34 FITC (Invitrogen), Streptavidine QDot 605 (Invitrogen).