Cd11b microbeads mouse human

Six- to eight-week-old female C57BL/6 mice were purchased from Charles River, housed and treated under the University of Pennsylvania Institutional Animal Care and Use Committee-approved protocols. SWV FRβ KO or WT mice were kindly provided by Richard H. Finnel (Baylor College of Medicine, Houston) and were breeded in-house^{96 (link)}. Six- to eight-week-old male and female SWV were used.
Mice were inoculated i.p. with 5 × 10⁶ ID8 RFP-fLuc and euthanized at indicated time points to collect tumor ascites by peritoneal wash. Ten milliliters of phosphate-buffered saline (PBS) were injected i.p. and total cells in the wash were collected. Red blood cells were lysed using ACK (ammonium-chloride-potassium) lysis buffer (Thermo Fisher). For flow cytometry assays, 1–2 × 10⁶ total cells were stained and analyzed as described below. For in vitro coculture assays, cells were labeled with CD11b MicroBeads (mouse/human) (Miltenyi Biotec) and isolated with LS MACS separation columns according to the manufacturer’s instructions. CD11b⁺ cells were stained for markers CD45 and F4/80, and live, double-positive cells were sorted based on FRβ expression by the Flow Cytometry and Cell Sorting Facility (UPENN).

Minced spleens and lymph nodes were passed through a 40μm strainer to get a single-cell suspension as described previously [16 (link), 47 (link)]. After lysis of red blood cells, the cell suspension was incubated with CD11b microbeads mouse / human (130-049-601, Miltenyi Biotec) for 15 minutes at 4°C. CD11b-positive cells were eluted from the beads using MACS^® separation columns (130-042-201, Miltenyi Biotec) according to the manufacturer’s instructions and collected for subsequent RNA extraction.

Randomly selected, de-identified ascite samples from ovarian cancer patients were purchased from the University of Pennsylvania Tumor BioTrust Collection, where ovarian cancer samples are collected under an IRB-approved research protocol (IRB702679). Written informed consent was obtained from each patient. Single-cell suspensions from liquid tumor ascites were labeled with antibodies and analyzed by flow cytometry. For in vitro assays, cells were labeled with CD11b MicroBeads (mouse/human) (Miltenyi Biotec) and isolated with LS MACS separation columns according to the manufacturer’s instructions. CD11b⁺ and CD11b⁻ fractions were cocultured with CAR-T cells for cytokine release and/or lytic assays as described above.