Gallios flow cytometry

Forty-eight hours after transfection, TPC1 and KTC1 cells were collected for cell cycle and apoptosis assay. For cell cycle analysis, cells were fixed in 75% ethanol overnight at 4 ℃. Cell Cycle and Apoptosis Analysis Kit (Beyotime biotechnology, Jiangsu, China) was applied to stain the treated cells according to the manufacturer's instruction. Then, the cells were analyzed by Gallios Flow Cytometry (Beckman Coulter, USA) in order to quantify the proportion of cells in each stage of cell cycle (S, G1, G2/M). For apoptosis assay, the Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit (Life technology) was used. The treated cells were suspended by 100 μl binding buffer with 5 μl Annexin V and 1μl propidium iodide, followed by 15 minutes' incubation protected from light. Then 400 μl binding buffer was added and the cells were resuspended. Gallios Flow Cytometry (Beckman Coulter, USA) was applied to quantify the proportion of cells marked with Annexin V and propidium iodide. All experiments were performed independently in triplicate.

Fourty-eight hours after transfection, U87 and U251 cells were collected for cell cycle and apoptosis analysis. The cell cycle analysis was performed using the Cell Cycle Analysis Kit (Beyotime biotechnology, Jiangsu, China) according to the manufacturer’s instructions. The apoptosis assay was performed using the Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit (Thermo Fisher Scientific, Waltham, MA, USA). The cells were detached by trypsinase, centrifuged at 1000 g for 5 min and then resuspended in 100 μL of binding buffer with 5 μL of Annexin v. After that, 1 μL of propidium iodide was added and mixed, followed by 15 min’ incubation in dark. Subsequently, 400 μL of binding buffer was added to resuspend the cells, which were analyzed by Gallios Flow Cytometry (Beckman Coulter, USA). The experiments were repeated triple times.

Immunofluorescence flow cytometry was performed as described previously^{3 (link)}. For mAb staining, the cells were washed with staining buffer (1% FBS in HBSS), resuspended in 50 μl of the same buffer, pre-incubated with purified anti-mouse CD16/32 (Biolegend) for 10 min, and incubated for 30 min at 4 ˚C with each fluorescence-conjugated mAb or isotype control matched with primary antibody. Zombie NIR™ dye (Biolegend) was used to assess live or dead status of cells. The samples were measured using a Gallios flow cytometry or CytoFLEX (Beckman Coulter). Doublets were distinguished from single cells by plotting FSC height vs FCS area. CD4⁺ T cells or Treg cells were sorted using a MoFlo XDP (Beckman Coulter). The purity of the sorted populations was more than 95%, as determined by a presorted sample run in parallel. Data were analyzed using in Kaluza analysis version 2.1 (Beckman Coulter).