Anti cd45 percp cy5

Stained cells were analyzed using LSR-Fortessa (BD Bioscience) and analysed with FlowJo software (Tree Star). The following antibodies were used to detect cell surface expression: anti-CD45-PerCPCy5.5, anti-CD11c-PeCy7, anti-Ly6C-FITC, anti-Ly6G-PE and anti-Ly6C-APC (BD Bioscience); anti-Ly6G-APC/Cy7 and anti-CD4-AF700 (Biolegend); anti-CD8-APC, anti-CD11b-PB, anti-IFNγ-PECy7 and c-MET-FITC (Invitrogen); anti-IFNγ-PE, anti-IL-4-FITC (eBioscience) and anti-Annexin V-PeCy7 (eBioscience).
Cell viability was analyzed using the Live/Dead fixable Aqua Dead Cell Stain Kit (Invitrogen) for ex vivo experiments, whereas DAPI (Sigma-Aldrich) was used for in vitro experiments. Parasite-infected cells were identifies based on the DsRed fluorescence of transgenic parasites.

PBMCs were isolated using Ficoll-Amidotrizoaat (Pharmacy LUMC) gradient centrifugation according to the standard operating procedure of the Medical Oncology department of LUMC. Isolated PBMCs were carefully resuspended and 3 times washed in PBS (B. Braun, Melsungen, Germany). Samples were fixed in 1.5% formaldehyde and permealized in ice-cold pure methanol. Cells were washed 3 times in staining buffer (PBS with 5% bovine serum albumin (BSA, Sigma, St Louis, USA)) and stained for 30 min on ice with anti-CD45-PerCP-Cy5.5 (BD Bioscience, Breda, the Netherlands), clone 2D1 anti-CD3-PE (BD, clone SK7), anti-CD14-AF700 (BD, clone M5E2), anti-CD15-PE CF594 (BD, clone W6D3) and anti-γ-H2AX-AF488 (Biolegend, clone 2F3), followed by another washing step and resuspension in PBS. Per experiment we used 1,000,000 cells or more when available. The cell acquisition was performed immediately after the staining procedure on the flow cytometer (BD LSR Fortessa Flow Cytometer analyzer, BD Bioscience, Breda, The Netherlands) and data were analyzed using BD FACS Diva Software version 6.2. The CD45+ cells were gated, after which the CD3+ T-lymphocytes, CD3− non-T cells (also harboring B lymphocytes) or CD14+ CD15− monocytes were analyzed for the geomean (as measure for the intensity) of γ-H2AX (Supplementary Fig. 2).

Expression of the cell surface markers CD73, CD90 and CD105 and absence of hematopoietic markers on MSCs was determined using flow cytometry, according to the statement by the International Society for Cellular Therapy [30 (link)]. Cells were detached using Trypsin/EDTA and washed in PBS/0.05% bovine serum albumin. Antibodies used were (FITC) conjugated anti-CD86-fluorescein isothiocyanate (FITC) (cat. no. 555657), anti-HLA-DR-FITC (cat. no. 347400), anti-CD31-phycoerythrin (PE) (cat no 555446), anti-CD34-PE (cat no 348057), anti-CD73-PE (cat no 550257), anti-CD90-PE (cat no 555596), anti-CD3-peridinin chlorophyll protein(PerCP)-Cy5.5 (cat no 332771), anti-CD45-PerCPCy5.5 (cat no 332784), all from BD (San Diego, CA, USA) and anti-CD105-PE (cat no SN6), from Ancell (Bayport, MN, USA). Flow cytometry was performed on a FACScalibur, and analyzed using Cellquest software (both Becton–Dickinson). Mean fluorescence intensity (MFI) ratio was calculated by determining the MFI of the specific staining relative to the MFI of the appropriate isotype control staining.

The following mAbs were used for cell surface staining: anti-CD45-PerCpCy5.5, anti-Ly6G(1A8)-PE or FITC, anti-Ly6C-APC or FITC (all from BD Bioscience), anti-CD11b-Pacific blue, anti-CD11c-PECy7, anti-CD4-AF700, anti-IL-4-FITC, and anti-IFN-γ-PECy7 (all from eBioscience); anti-CD3-PE or APC and anti-IL-10-PE (Biolegend); and anti-CD8-APC (Invitrogen). For the exclusion of dead cells, we used the Live/Dead fixable Aqua Dead Cell Stain Kit (Invitrogen). L. major mCherry was detected using Texas red as a positive control, L. mexicana DsRed and L. major Sd-RFP were detected using PE as a positive control. Fluorescent parasites and stained murine cells were acquired using a flow cytometry analyzer, either BD LSRII or BD LSR-Fortessa Series (Beckton Dickinson) machines, the data was acquired with the DIVA software and the results were analyzed with FlowJo 10 software.

Serum immunoglobulins (Ig) levels (IgG, IgA and IgM) were measured by immunoturbidimetry using the automatic analyzer Alinity c system (Abbott Laboratories, Chicago, IL, USA). For cellular evaluation, EDTA whole blood samples were collected. Lymphocyte subpopulations (CD4+ T, CD8+ T, B and NK cells) were performed using BD Trucount tubes and the BD Multitest 6 Color BTNK kit (BD Biosciences, San Diego, CA, USA), which included the following mixtures of fluorophore-conjugated monoclonal antibodies (mAb): anti-CD45-PerCP-Cy5.5, anti-CD3-FITC, anti-CD8-APC-Cy7, anti-CD4-PE-Cy7, anti-CD19-APC, and anti-CD16+CD56-PE. B cell phenotype was performed with an eight-color panel of the following mAb: anti-CD45-APC-H7, anti-CD19-V500, anti-CD10-V450, anti-CD38-PE-Cy7, anti-CD21-PE, anti-CD27-PerCP-Cy5, anti-IgD-FITC, and anti-IgM-APC (BD Biosciences, San Diego, CA, USA), following EURO-Class classification. Cells were acquired on a BD FACSCanto II Flow Cytometer (BD Biosciences, San Diego, CA, USA), and the InfinicytTM22.0 software was employed for multiparametric analysis (Cytognos SL, Salamanca, Spain).