Cd16 cd32 mouse fc block

Prior to tissue harvest, animals were anesthetized with isofluorane and intracardially perfused with cold heparinized saline. After removing the cerebellum and olfactory bulbs, brain tissue was processed immediately by fine cutting followed by digestion with an enzyme cocktail of collagenase/DNase (Sigma). Myelin was removed by density centrifugation in DMEM supplemented with 20% BSA, and the remaining cell pellet was resuspended in media for staining. Brains were processed individually, without pooling. Isolated cells were first blocked using CD16/CD32 Mouse BD Fc Block (BD Biosciences, San Jose, CA), and surface proteins were detected using monoclonal antibodies CD45 FITC (30-F11, Biolegend, San Diego, CA), CD11b BV510 (M1/70, Biolegend), CCR2 BV650 (SA203G11, Biolegend), CD3e PE (145-2C11, BD Pharmingen), CD19 PE-Cy7 (6D5, Biolegend), CD11c APC (N418, Biolegend), and Ly6c APC-Fire750 (HK1.4, Biolegend), CD74 BV421 (In1, BD). Dead cells were excluded using Live/Dead Fixable Blue (Thermo Fisher Scientific, Waltham, MA), according to manufacturer’s instructions. After staining, cells were washed and acquired using a FACSymphony (BD Scientific) equipped with FACS Diva v.8, and data were analyzed using FlowJo software v.10.0.7.

To analyze CSC phenotypes, TU-BcX-4IC was 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 PBS. Collected cells from the tumor and blood samples were placed in a staining solution containing 1% Bovine Serum Albumin (BSA; Sigma-Aldrich) and 1% CD16/CD32 Mouse BD Fc Block™ (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 5,000 events were analyzed and reported as the mean ± SEM.

Epithelial cells from WT, LApc, and LApcL mice and stromal cells from Apc^Min and VApc^MinL mice were sorted on BD Influx (BD Biosciences) for scRNA-seq analysis and gene expression validation. The epithelial cells were surface-stained with the following antibodies at 1:500 dilution for 30 min on ice: anti-Mo CD326 (EpCAM) phycoerythrin (eBioscience; Invitrogen, #12-5791-82), CD16/CD32 (mouse BD Fc block; BD Pharmingen, #553141), anti-CD45 (eBioscience, #48-0451-82), and anti–Ter-119 (eBioscience, #48-5921-82). Sorted cells were resuspended into Hanks’ balanced salt solution with 0.04% BSA.

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).

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 analyze CSC phenotypes, TU-BcX-2O0 was 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 0.5M EDTA (Gibco Invitrogen, Carlsbad CA, USA), incubated in red blood cell lysis buffer (0.008% NH4Cl, pH 7.2-7.4; Sigma-Aldrich, St. Louis MO, USA) 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), anti-human CD326 (EpCAM; PerCP-eFluor710) 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.