Anti mouse cd45 percp cy5

After 2-week culture in α-MEM plus 10% FBS, mouse BMSCs were trypsinized, pelleted and then resuspended in PBS containing 2% FBS to a concentration of 2 × 10⁶ cells/ml. Freshly isolated bone marrow cells were resuspended to a concentration no more than 1 × 10⁷ cells/ml. Cells were stained with anti-mouse CD45 PerCP-Cy5.5 (eBioscience, 45-0451-80), anti-mouse/rat CD29 PE-Cy7 (eBioscience, 25-0291-80), anti-mouse CD105 (Endoglin) PE (eBioscience, 12-1051-81), and anti-mouse Ly-6A/E (Sca-1) APC (eBioscience, 17-5981-81), and analyzed with an LSRII cytometer (Becton Dickinson, San Jose, CA, USA). For BrdU Incorporation, the mice were intraperitoneally labeled twice (a 16-h interval) with 1 mg/mouse/injection BrdU and euthanized 2 h after the second injection. Freshly harvested bone marrow was treated with 1× RBC Lysis Buffer (eBiosciences, San Diego, CA, USA). The remaining cells were stained with anti-mouse CD45 PerCP-Cy5.5, anti-mouse/rat CD29 PE-Cy7, anti-mouse CD105 (Endoglin) PE, and anti-mouse Ly-6A/E (Sca-1) Alexa Fluor 700 (eBioscience, 56-5981-82) before permeabilization, DNase treatment and staining with anti-BrdU APC according to the instruction of APC BrdU Flow Kits (BD Pharmagen, Franklin Lakes, NJ, USA). The percentage of BrdU-positive fractions was assessed using FlowJo analysis software (Tree Star, Ashland, OR, USA).

Cells from in vivo digested lymph nodes and lungs were dissociated as previously described and stained with live/dead reactive dye, anti-mouse CD45 PerCP-Cy5.5 (eBioscience, Cat. # 45-0451-82), anti-mouse CD31 FITC (eBioscience, Cat. # 11-0311-82), and anti-mouse gp38 PE-Cy7 (eBioscience, Cat. # 25-5381-82). Flow cytometry samples were processed using the Millipore Guava easyCyte 8HT Flow Cytometer and analyzed using InCyte software for total LEC counts per mg as well as percentage of LECs. Gating was done first on live cells, followed by CD45- populations. This subpopulation was gated for gp38 and CD31 with the following populations: CD31+gp38+ (LECs); CD31+gp38-(blood endothelial cells), and CD31-gp38+(fibroblastic reticular cells).

Mice were administered 1mg of bromodeoxyuridine (BrdU) via intraperitoneal (i.p.) injection at 24 h, 16 h, and 1 h prior to harvest of tumors and spleens. Minced tumor slurries were dissociated in media containing collagenase IV (400 units/ml; Worthington) and DNase I (100 μg/ml; Roche). Single-cell suspensions were prepared from spleens and labeled with an antibody cocktail containing anti-mouse CD45-PerCP-Cy5.5, anti-mouse CD8-Pacific Blue, anti-mouse CD4-Alexa Fluor 750, and anti-mouse CD49b-APC (eBioscience). Intracellular antibody staining against BrdU was performed using a commercially available kit (BD) according to the manufacturer's instructions. Data were acquired on a BD LSR II flow cytometer and analyzed using FACS Diva software.