Anti ly6c pe

Mice were sacrificed at 1 day and 7 days after MI. The blood, spleen, bone marrow and infarcted myocardium were collected and made into single-cell suspensions for flow cytometry, as previously described26 (link)52 (link). The cells were stained with a mixture of antibodies (anti-CD11b-APC, anti-Gr-1-PerCP-Cy5.5, anti-Ly6C-PE; BD Biosciences). Data were acquired using an LSRII flow cytometer (BD Biosciences) and were analyzed with FlowJo7 software (Tree Star, Inc.).

Splenocytes obtained from control or treated tumor bearing mice were liquid-nitrogen frozen and thawed before tests. Then, for lymphoid cell analysis, they were stained in a one-step test with the following fluorophore-labeled anti-mouse monoclonal antibodies (mAbs): anti-CD4-APC (BD Pharmingen, USA, RM4-5), anti-CD8-PE-Cy7 (BD Pharmingen, USA, 53–6.7), anti-CD49b-PE (BD Pharmingen, USA, DX5) and anti-CD19-FITC (BD Pharmingen, USA, 1D3). Phenotype analysis was carried out using the Becton Dickinson FACSCalibur apparatus with Cell Quest Software. For myeloid cell characteristics, the flow cytometry analysis of MDSC surface phenotype was performed as described previously [16 (link)] using fluorophore-labeled anti-mouse mAbs: anti-CD11b-PerCP-Cy5.5 (BD Pharmingen, USA, M1/70), anti-B220-APC (BD Pharmingen, USA, RA3-6B2), anti-Ly6G-APC-Cy7 (BD Pharmingen, USA, 1A8), anti-Ly6C-PE (BD Pharmingen, USA, AL-21) and anti-MHCII-FITC (BD Pharmingen, USA, 25-9-17). The cells were stained for 45 min at 4°C. The viability of spleen cells was assessed by incubation with DAPI dye. The analysis was carried out using Becton Dickinson FACSFortessa apparatus with FACSDiva software.