Facscelesta analyzer

Flow cytometry analysis was performed to assess the cellular composition of individual PDX lines and evaluate the purity and yield of human tumor cells isolated from each PDX tumor. Cells obtained from the PDX dissociation procedure were suspended in 80 μL of MACS buffer (PBS pH 7.2, 0.5% BSA, 2mM EDTA) and plated at a density of 0.5 × 10⁶ cells per well in 96-well plate. Next, 20 μL of human FcR blocking reagent (Miltenyi Biotec, #130-059-901) was added to the cells and the cells were incubated for 5 min at 4 °C. Following the incubation and washing of cells, a human-specific antibody, CD326 (EpCAM)-PE (Miltenyi Biotec, #130-113-264, dilution 1:11) for detection of carcinoma cells and/or a Labeling Check Reagent-APC (Miltenyi Biotec, #130-122-219, dilution 1:4) for the detection of mouse cells labeled with MACS MicroBeads, was added to the respective wells and incubated for 10 min at 4 °C. Finally, the cells were washed, resuspended in 300 μL of MACS buffer, and processed using a BD FacsCelesta analyzer (BD Biosciences, San Jose, CA, USA) according to manufacturer’s protocol. The flow cytometry data was analyzed using FlowJo software v10 [22 (link)].

To assess the effects on mitochondrial transfer, 1 × 10⁷ tenocytes were labeled with CellTracker™ Violet (CTV, Invitrogen) to distinguish them from MSCs in the co-culture system. CTV-positive cells were sorted using a FACSAria III sorter (BD Biosciences). Following cell sorting, the tenocytes were evaluated for apoptosis, oxidative stress, and mitochondrial function.
Tenocyte proliferation was assessed using flow cytometry (BD FACSCelesta) to determine the number of CTV-positive cells. Apoptosis was detected using an Annexin V-FITC Apoptosis Detection Kit (Beyotime). Briefly, sorted cells were harvested, re-suspended in 195 μL annexin V binding, and stained with 5 μL Annexin V-FITC and 10 μL PI for 10 min at 22–26 °C. A BD FACSCelesta analyzer was used to detect the distribution of cell populations in different quadrants.

Following T cell transfer, four 3 mm punch biopsies from distal colon were collected. Biopsies were weighed and cultured in 48-well plates with 500 μL RPMI containing 10% FBS for 6 hours. Supernatants were collected and analyzed using a cytometric bead array (CBA) and the mouse Th1/Th2/Th17 cytokine kit (BD Biosciences) and analyzed using a BD FACS Celesta analyzer and FCAP array software v3.0.