Anti cd11b fitc

Cells from subconfluent monolayer cultures were suspended in phosphate-buffered saline (PBS) and incubated with monoclonal anti-human ALCAM-phycoerythrin (PE) (R&D Systems, Minneapolis, MN), anti-human CD24-fluorescein isothiocyanate (FITC) (eBioscience Inc, San Diego, CA), anti-human CD44-FITC (MBL, Nagoya, Japan), and monoclonal anti-human CD133-FITC (Ancell Corp, Bayport, MN) antibodies. Mouse immunoglobulin G1 K isotype Control PE (eBioscience Inc) was used as a negative control. Mouse IgG1 K isotype Control PE and anti-CD11b-FITC (Miltenyi Biotec) were used to exclude mouse cells from analyses. Labeled cells were analyzed by a flow cytometer (EC800; Sony Biotechnology, Tokyo, Japan). For cell separation, we incubated magnetic microbeads conjugated with anti-PE reagent (Miltenyi Biotec) with labeled cells for 15 minutes at 4°C. Labeled cells were isolated by passing the suspension through an AutoMACS PRO separator (Miltenyi Biotec). Unlabeled cells were negatively selected and collected by the depletion method through the AutoMACS PRO separator.

Brains from 22- to 26-week-old animals were homogenized using a 5-ml Glass Tissue Grinder (A. Hartenstein). The homogenized solution was mixed with 100 % Percoll solution (GE healthcare) to a final 70 % Percoll solution and layered under a 30 % Percoll solution. After 30 min centrifugation at 500g without brake at RT, the cells were isolated from the 30–70 % layer and washed with PBS. Per staining, 100 000 cells were used. The staining was performed in FACS-PBS (PBS, 2 % FCS, 5 mM EDTA) in the presence of the FcBlocker (Miltenyi Biotec) using the following fluorescently labeled antibodies: anti-CD4-PE, anti-CD8-APC, anti-CD11b-FITC, and anti-CD45-APC (all from Miltenyi Biotec), for 15 min at RT in the dark. Samples were analyzed using a Gallios flow cytometer (Beckman Coulter). Thirty thousand events were recorded. Flow cytometry data were analyzed using the FlowJo 8.5.3 software (FlowJo, LLC). For viability testing, the Fixable Viability Dye eFluor^® 780 (eBioscience) was used according to the manufacturer’s instructions. The gating of the living cell population was performed based on the live staining; dead cell populations were excluded. The total cell number was determined utilizing bead measurement (Beckman Coulter CC Size Standard L10), and the absolute cell numbers were calculated according to the manufacturer’s instructions.

In order to characterize the phenotype of splenocytes (SPC) and tumor-infiltrating immune cells, single-cell suspensions were derived from the spleens and from lungs of wt tumor-bearing and K-ras^G12D-vaccinated mice, as described in [73 (link)]. Cells were treated with Fc receptor blocker (BD Biosciences) and then stained for 30 min at 4 °C with the following antibodies: anti-CD45-VioGreen, anti-CD3-FITC, anti-CD4-APC/Vio770, anti-CD8-VioBlue, anti-CD49b-PE, anti-TCRγδ-PE/Vio770, anti-PD1-APC, anti-CD11b-FITC, anti-F4/80-PE/Vio770, anti-Ly6C-APC/Vio770, anti-Ly6G-VioBlue, anti-MHCII-APC (Miltenyi Biotec Bergisch Gladbach, Germany), anti-CD206-PE, and anti-CD69-PE/Vio770 (Biolegend, San Diego, CA, USA). Labeled samples were acquired on a BD FACSVerse and analyzed using FlowJO10.5.3 software.