Apc cy7 rat anti mouse cd45

Overnight culture of YFP-PAO1 was re-suspended in antibiotic-free DMEM medium containing 10% heat-inactivated FBS. J774A.1-CTR or J774A.1-EZR cell lines were seeded in 3.3 cm dishes (1 × 10⁶) and incubated with YFP-PAO1 at an MOI bacteria:cells of 100:1. After 30 min, phagocytosis was stopped by transferring cells to ice. Cells were washed in PBS, incubated with Fc block (BD Pharmingen) for 5 min on ice, and labeled with rat anti-mouse CD45-APC-Cy7 (Clone:30-F11, BD Pharmingen) for 30 min. All samples were acquired on an ImageStream®X Mark II using 488 and 642 nm laser. A total of 10000 events were collected for each sample. The IDEAS software (Amnis Corporation, Seattle Wa) was used to assess the phagocytosis index. For details of the ImageStream gating strategy (Fig. 3D), please refer to the online supplemental experimental procedures. Phagocytosis was further confirmed by colony-forming unit (CFU) assay (PAO1 at an MOI of 20:1), performed using standard procedure described in the online supplemental experimental procedures.

FACS analyses were performed on BD FACSDiva Cell Sorter (BD FACSDiva Software, RRID:SCR_001456) at the Duke Flow Cytometry Core. For isolation of SP and non-SP cells, single-cell suspensions were treated with 2.5 mg/mL Hoechst 33342 dye (MilliporeSigma, B2261) alone, or in combination with 50 mmol/L verapamil (MilliporeSigma, V4629) as a negative control, for 90 minutes at 37°C. SP cells were identified using dual-wavelength analysis (blue, 424–444 nm; red, 675 nm) after excitation with 360 nm UV laser. To enrich for tumor SP cells, digested tumor mixture was stained with rat anti-mouse CD45-PE-Cy7 (BD Biosciences, 552848, RRID:AB_394489) or rat anti-mouse CD45-APC-Cy7 (BD Biosciences, 557659, RRID:AB_396774) antibody at 1:800 dilution, or cells were sorted on the expressed fluorescent reporters. RFP and YFP cells from the KPCC tumors were identified using the blue 488 nm laser. Dead cells were eliminated with propidium iodide (Thermo Fisher Scientific), according to the manufacturer’s instructions. To analyze the purity of the sorted cells, we took a small sample of sorted cells immediately after FACS and reanalyzed for the presence of different sorted fractions using the same sorter (FACSDiva Cell Sorter, RRID:SCR_001456). Analysis of flow cytometry data were performed using FloJo (version 10.7).