Lsrii flow cytometry system

After 48 h transfection, UM-UC-3 and T24 cells were washed with PBS and fixed in 75% ethanol at ?20°C. After 24 h fixation, the cells were washed with PBS and treated with DNA Prep Stain (Beckman Coulter, Fullerton, CA) for 30 min. Cell cycle analysis was conducted by BD LSRII Flow Cytometry System with FACSDiva software (BD Bioscience, Franklin Lakes, USA). The cell cycle distribution was illustrated as the percentage of cells in G1, S, and G2 populations and data was evaluated by ModFit LT software package.

48 h after the transfection of RNA duplexes (50 nM of NC, miR-608 or siFLOT1) or RNA oligos (100 nM of inhibitor NC or miR-608 inhibitor), or after the co-transfection of RNA duplexes and RNA oligos (FLOT1 rescue experiment), bladder cancer cells were harvested and washed with PBS and fixed with 75% ethanol at -20 °C. After 24 h fixation, the cells were washed with PBS again and stained with propidium iodide using the cell cycle and apoptosis analysis kit (Beyotime, China) for 30 min. Cell cycle features were analyzed by BD LSRII Flow cytometry system with FACSDiva software (BD Bioscience, USA). The data were analyzed by ModFit LT 3.2 software (Verity Software House, USA).

PIGAwt, PIGAc.1234C>T and PIGAtr411 hiPSCs were stained with an Allophycocyanin (APC)-conjugated anti-human CD59 antibody (NB500-400APC, clone MEM-43/5, Novus Biologicals). CD59 is a GPI anchored protein and was used as an indicator of functional GPI anchor production. Cells were incubated with antibody on ice for 30 minutes and analyzed by flow cytometry (BD Biosciences). Hematopoietic cells were stained with APC-conjugated mouse anti-human CD34 (555824, BD Biosciences), PE-conjugated mouse anti-human CD45 (555483, BD Biosciences), FITC-conjugated mouse anti-human CD15 (11-0159-42, eBioscience), PE-conjugated mouse anti-human CD33 (555450, BD Biosciences), FITC-conjugated mouse anti-human CD71 (555536, BD Biosciences), and FITC-conjugated mouse anti-human CD235a (559943, BD Biosciences) by incubating on ice in the dark for 30 minutes and analyzed by flow cytometry (BD LSRII flow cytometry system, BD Biosciences). Samples were gated based on physical parameters (forward and side scatter). Gating for FITC-conjugated and APC-conjugated antibodies was based on the isotypic control antibodies and was confirmed by reference to negative controls. FACS data was analyzed using FlowJo Software (Treestar Inc.).

Cells were harvested 72 h after the RNA transfection. After washed with PBS, the cells were fixed in 75% ethanol overnight at −20°C. Then the cells were washed with PBS for twice and applied with DNA Prep Stain (Beckman Coulter, Fullerton, CA, USA) and RNase for 30 min. Next, the cell cycle analysis was performed by BD LSRII Flow Cytometry System with FACSDiva software (BD Bioscience, Franklin Lakes, USA). The data were analyzed by ModFit LT 3.2 software (Verity Software House, Topsham, USA) and the cell cycle distribution was described as the percentage of cells in G1, S, and G2 populations.

A total of 7.5 x 10⁴ cells/well were plated in triplicate in 6 well plates and grown for 1–7 days. Cells were incubated with complete medium as previously described. The number of cells was counted at the indicated time-points in triplicate.
For flow cytometry analysis, cells were plated at a density of 1 x 10⁵ cells/ml. The cells were pulse-labeled with BrdU for 30 min, washed with PBS, and treated with trypsin. Cells were fixed with ethanol and stained with anti-BrdU-FITC antibody (BD Biosciences) and propidium iodide, according to the manufacturer’s instructions. Flow cytometry analysis was carried out with the BD LSRII flow cytometry system and BD FACSDiva software.