Anti cd4 pe

For lymphocytes differentiation, blood samples were harvested at indicated time points and monocytes were separated though isodensity centrifugation. Thereafter, approximately 10⁶ cells were re suspended in 100 μL of PBS and incubated with 2 μg anti-CD3c FITC together with anti-CD4 PE or anti-CD8 PE antibodies (all from BioLegend, San Diego, CA, USA) for 20 min on ice. Thereafter, the cells were washed twice with PBS and flow cytometry was performed using a FACSort instrument (BD Biosciences, San Jose, CA, USA). Data was analyzed using FlowJo software (Tree Star, San Caros, CA, USA).

After the cells had been prepared, between 500,000 and 1.25 million cells from these single-cell suspensions were incubated with 1 μg of the appropriate primary antibodies (listed below) for 45 minutes on ice, with vortexing at every 7–10 minute intervals. These cells were washed twice with phosphate buffer followed by incubation for 15 minutes with an appropriate second step reagent-conjugated to a fluorochrome. After 15 minutes the cells were washed twice with PBS, and re-suspended in 1XPBS and fixed with paraformaldehyde at 1% final concentration. 10,000 and 25,000 cells were analyzed with a Becton Dickinson FACS Calibur (BD Biosciences, San Jose, CA, USA). Primary antibodies used in this investigation were: Anti-CD3-FITC, anti-CD3-PE anti-CD4-FITC, anti-CD4-PE, anti-CD44 Biotin, anti-B220-FITC and anti-IgM-PE (BioLegend Inc., San Diego, CA, USA). Also anti-CD25-PE, anti-CD5-PE, anti-IgM-PE, anti-IgA-FITC, and anti-B220-PerCP (BD Biosciences, San Jose, CA, USA). Secondary antibodies used were: Streptavidin-PErCP (BioLegend Inc, San Diego, CA, USA) and Streptavidin-PE (BD Biosciences, San Jose, CA, USA).

The following antibodies were purchased from Biolegend Inc. (San Diego, CA, USA): anti-CD3-pacific blue (17A2), anti-CD4-pacific blue (GK1.5), anti-NK1.1-pacific blue (PK136), anti-CD11b-pacific blue (M1/70), anti-CD11C-pacific blue (N418), anti-Ter-119-pacific blue (Ter-119), anti-CD8-pacific blue (53-6.7), anti-B220-pacific blue (RA3-61B2), anti-CD4-PE (GK1.5), anti-CD4-FITC (GK1.5), anti-CD8-APC (53-6.7), anti-CD8-Pecy7 (53-6.7), anti-CD23-PECy7 (B3B4), anti-CD21/CD35 (CR2/CR)-PerCP/Cy5.5 (7E9), anti-Gr1-Pecy7 (RB6-8C5), Anti-IAb-FITC (KH74), anti-B220-APC-Cy7 (RA3-61B2), anti-CD117-PE-Cy7 (2B8), anti-PDCA1-APC (927), anti-CD135 (flt3)-APC (A2F10), anti-CD25-PE (3C7), anti-CD11C (N418), anti-IgD-PerCP/Cy5.5 (11-26c.2a), anti-Sca1-APC (D7), anti-IgM-PE (RMM-1), anti-CD16/32-APC/Cy7(93), anti-CD127 (IL-7R)-PerCP/Cy5.5 (SB/199), anti-CD93 (AA4.1)-PE (AA4.1), anti-CD117 (c-kit)-PE/Cy7 (2B8), and anti-CD34-PE (MEC14.7). Anti-human/mouse phospho-ERK1/2 (T202/Y204)-APC (MILAN8R), anti-human-mouse phospho-p38 (T180/Y182)-APC (4NIT4KK), and anti-human/mouse phosphor-AKT (S473)-APC (SDRNR) were purchased from eBioscience (San Diego, CA, USA).

Single cell suspensions from murine primary pancreatic tumors and pulmonary metastasis were prepared by mechanical and enzymatic disruption and tumor cells, tumor associated macrophages and stromal cells were analyzed and sorted using flow cytometry (FACS ARIA II, BD Bioscience). Samples were digested as outlined above, the cells were then filtered through a 70 μm cell strainer and resuspended in PBS + 1% BSA, blocked for 10 min on ice with FC Block (BD Pharmingen, Clone 2.4G2) and stained with Sytox® blue viability marker (Life Technologies) and conjugated antibodies anti-CD45-PE/Cy7 (Biolegend, clone 30-F11) and anti-F4/80-APC (Biolegend, clone BM8).
Blood was collected from mice via tail vein bleed in EDTA-tubes. Red blood cell lysis was performed and resulting leukocytes were resuspended in PBS + 1% BSA and blocked for 10 min on ice with FC Block and stained with Sytox® blue viability marker and conjugated antibodies anti-CD45-APC/Cy7 (Biolegend, 103115), anti-CD11b-APC (Biolegend, 101212), anti-Ly6G-PerCP-Cy5.5 (Biolegend, 127616), anti-Ly6C-PE (Biolegend, 128008), anti-CD3-PE-Cy7 (Biolegend, 100320), anti-CD4-PE (Biolegend, 100408), and anti-CD8-PerCP-Cy5.5 (Biolegend, 100734). Cell analysis was performed using FACS Canto II.