Lsr 2 green flow cytometer

Side population (SP) of putative CSCs were performed as previously described [18 (link), 19 (link)]. Briefly, 1 × 10⁶ cells/ml of human PC or syngeneic-derived cancer cells were washed with PBS, suspended in DMEM containing 10% FBS, and incubated with 2 μg/ ml of Hoescht 33342 (AnaSpec Inc., Fremont, CA, USA) fluorescent dye for 90 minutes at 37°C in the dark with intermittent shaking for 90 minutes at 37°C. Cells were incubated with 100 μM of Verapamil (Sigma, St Louis, MO, USA) for 20 minutes at 37°C with intermittent shaking prior to Hoescht 33342 staining. After incubation, cells were immediately washed thrice with cold PBS and resuspended for counterstaining with 1 mL of culture media containing 10 μg/ml of propidium iodide to label dead cells. After gating, SP and non-SP cells were analyzed using BD LSR II Green flow cytometer (BD Biosciences, San Jose, CA).

To analyze cellular biodistribution of P-Tofa, Alexa Flour^® 647 labeled P-Tofa (P-Tofa-Alexa 647 200 mg polymer/kg mouse) was mixed with P-Tofa to a total Tofa equivalent dose of 210 mg/kg and i.v. injected to mice with DSS exposure (n = 3/time point) on day 7 post-induction. Mice were euthanized 1 and 3 days after the administration of P-Tofa-Alexa 647. Blood and major organs (liver, spleen, kidney, colon, and mesenchymal lymph nodes) were collected and processed for flow cytometry analysis. Blood cells were marked by the following antibodies: BF650-labeled anti-mouse F4/80, BV711-labeled anti-mouse CD11b, BV786-labeled anti-mouse CD3e, and PE-Cy7-labeled CD19; BV421-labeled anti-mouse CD11c, APC-eFluor 780-labeled anti-mouse Ly-6G (BD Biosciences), PE/Dazzle™ 594 anti-mouse CD45 (Biolegend). The cells were analyzed using LSR II Green flow cytometer (BD Biosciences) and FlowJo software (Treestar, Inc., San Carlos, CA).

Animals were euthanized and spleens were isolated. Single cell suspension of 1×10⁶ splenocytes in 100 μL DPBS was stained with a rat anti-mouse CD4-PE monoclonal antibody (eBioscience, San Diego, CA, USA). Cells were analyzed using an LSRII Green flow cytometer (BD Biosciences, San Jose, CA, USA) for CD4-PE and Foxp3-GFP and data were analyzed with FlowJo software (FlowJo, LLC., Ashland, OR, USA).

Single-cell suspensions were blocked with CD16/32 Fc blocking antibody (Cat# 101320, Biolegend) for 15 min and incubated with antibodies against surface markers for 30 min at 4°C, followed by 3 washes in FACS buffer. These samples were then run on an LSR II Green flow cytometer (BD Biosciences) and analyzed by FlowJo software (BD Biosciences).
Intracellular transcription factor staining was performed after surface staining. The cells were fixed with buffer from the True-Nuclear Transcription Factor Buffer Set (Cat# 424401, BioLegend) for one hour according to the manufacturer’s protocol. Cells were then washed with permeabilization buffer and stained with antibodies against transcription factors in permeabilization buffer.
For intracellular cytokine staining, single-cell suspensions were stimulated with anti-CD3/CD28 (Cat# 100340 and 102116, respectively, Biolegend) in the presence of brefeldin A (Cat# 420601, BioLegend) and 10 ng/mL IL
2 (Cat# 212–12, Peprotech) for 6 hours at 37°C. Subsequently, cells were collected for staining of surface markers and fixed by fixation buffer. Cells were then washed with permeabilization buffer and stained for cytokine-specific antibodies in permeabilization buffer.
All antibodies used for flow cytometry are listed in Supplemental Table 1.