Mouse igg1 fitc

Cells were trypsinized and washed with PBS x1, then fixed with 66% ethanol for 2 hours. After fixation, cells were again washed with PBS x1 then stained with propidium iodide (Abcam, Cambridge, UK) by resuspending the cells in 200 μl propidium iodide and RNase staining solution, as per the manufacturer’s protocol. Flow cytometry was performed using an Accuri-C6 flow cytometer. Cell cycle analysis was performed using the ModFit program to determine the percentage of cells in each of the cell cycle phases, for each cell line at each time point (ModFit settings were manual mode, excluding aggregates and debris, with a trapezoid S-phase shape). A FITC pre-conjugated CD44 antibody (CD44-FITC, Miltenyi Biotech, San Diego, CA, USA) was used to stain and quantify CD44+ cells, according to the manufacturer’s instructions. Isotype control antibody Mouse IgG1-FITC (Miltenyi Biotech, San Diego, CA, USA) was used to control for non-specific binding of antibody to cells.

Cells from the sorting procedure were centrifuged at 300g for 10 min and pellets resuspended in the appropriate fluorescent antibodies or isotype control antibodies at a concentration of 1:11 in MACS buffer. Cells were incubated in up to three antibodies for 10 min at 4 °C. After a final wash step, cells were resuspended in MACS buffer and transported directly for flow cytometry and analysed for surface marker expression using a Cyan ADP flow cytometer (Beckman Coulter, High Wycombe, UK) at the Faculty of Biology, Medicine and Health Core Facility, University of Manchester. Compensation was carried out using a bead kit (Miltenyi Biotec, Surrey, UK, 130-097-900). All antibodies and isotype controls used were obtained from Miltenyi Biotech (Surrey, UK) and are as follows with product codes: CD29-PE (130-101-275), CD34-PE-Vio770 (130-100-844), CD45-PerCP (130-098-145), CD90-FITC (130-097-930), CD146-VioBlue (130-099-678), CD271-APC (130-091-884), Mouse IgG1-PE (130-098-845), Mouse IgG2a-PE-Vio770 (130-098-564), Mouse IgG2a-PerCP (130-099-190), Mouse IgG1-FITC (130-098-847), Mouse IgG1-VioBlue (130-099-756), and Mouse IgG1-APC (130-098-846). Data were analysed using FlowJo v10 (FlowJo LLC, Ashland, OR, USA).