Galios flow cytometer

When PDX tumors were passaged from one mouse to the next, both the cells within the tumors and circulating cells in the peripheral blood of the PDX-implanted mice were analyzed for immune and CSC markers. TU-BcX-2 K1 tumors were enzymatically digested with type I collagenase (Worthington Biochemical Corporation, Lakewood, NJ, USA) at room temperature, neutralized with media, and then filtered. Circulating tumor cells were collected in whole blood with 0.5 M EDTA (Gibco Invitrogen, Carlsbad CA), incubated in red blood cell lysis buffer (0.008% NH4Cl, pH 7.2–7.4; Sigma-Aldrich, St. Louis MO) and washed with phosphate buffered saline (PBS). Isolated, harvested cells from the tumor and blood samples were placed in staining solution containing 1% Bovine Serum Albumin (Sigma-Aldrich) and 1% CD16/CD32 Mouse BD Fc BlockTM (BD Biosciences) in PBS. Anti-human CD24 (APC), anti-human CD326 (EpCAM; PerCP-eFluor710) and anti-human/mouse CD44 (PE-Fluor 610) primary antibodies were purchased from eBiosciences (San Diego, CA, USA). Cell populations from the tumors and blood were analyzed using a Galios Flow Cytometer (Beckman Coulter, Brea, CA, USA) running Kaluza software (Beckman Coulter). At least 5000 events were analyzed and reported as the mean ± standard error of mean (SEM).

MDDCs were plated at a density of 2x10⁶ per well in a 6-well plate. They were incubated alone or with Lactobacillus rhamnosus JB-1 or Lactobacillus murinus at 37°C, 5% CO₂ for 4 hours to 24 hours. Bacteria were added at a 1:1 ratio with MDDCs. After the incubation period, MDDCs were washed with PBS, harvested and incubation with pHrodo Green E. coli BioParticles Conjugate for Phagocytosis (Molecular Probes) was performed. Flow cytometry was performed using Galios flow cytometer (Beckman Coulter). Kaluza software (Beckman Coulter) was used for the analysis of pHrodo Green E. coli BioParticles Conjugate positive cells.

Circulating tumor cells were collected in whole blood with 0.5M EDTA (Gibco Invitrogen, Carlsbad CA), incubated in red blood cell lysis buffer (0.008% NH4Cl, pH 7.2–7.4; Sigma-Aldrich, St. Louis MO), and washed with PBS. Collected cells from the tumor and blood samples were placed in staining solution containing 1% Bovine Serum Albumin (BSA; Sigma-Aldrich) and 1% CD16/CD32 Mouse BD Fc BlockTM (BD Biosciences) in PBS. The following primary antibodies were used: Anti-human CD24 (APC), and anti-human/mouse CD44 (PE-Fluor 610) purchased from eBiosciences (San Diego, CA, USA). All cells from the blood were analyzed with a Galios Flow Cytometer (Beckman Coulter, Brea, CA, USA) running Kaluza software (Beckman Coulter). At least 5000 events were analyzed and reported as the mean ± SEM.

To determine the ratio of EGFP-positive cells, mES cells transfected with the EGFP gene by recombinase or integrase were collected, stained with DRAQ7 dye (Beckman Coulter, USA) to remove inactive cells, and then analyzed by a Galios flow cytometer (Beckman Coulter).