Annexin v 7aad apoptosis kit

For apoptosis studies, CCD-18Co cells were infected with lentivirus expressing shRNAs to either WNT2 or WNT5A or a non-targeting control shRNA, stained with a far-red tracking dye and mixed in a 50:1 ratio with HCT116-eGFP cells as previously described. After 72 hours of spheroid formation, spheroids were dissociated using non-enzymatic cell dissociation buffer (Gibco), stained with the Annexin-V/7-AAD Apoptosis kit (BD Biosciences) and subjected to flow cytometric analysis on a FACSCalibur (BD Biosciences). Cell quantitation was performed using FlowJo V10 software.
For WNT reporter studies, HCT116 cells were engineered to express a Super-Topflash (STF) luciferase reporter which is activated by the β-catenin-T-cell factor (TCF) transcriptional complex and is responsive to WNT signaling [52 (link)]. HCT116-STF cells were grown as monoculture spheroids or co-culture spheroids with CCD-18Co colon fibroblast cells. Spheroids were formed for 72 hours before treatment. Treatments included rhWnt5a protein (RnD Systems) at 25 nM or the porcupine inhibitor GNF-1331 at 100 nM. After 24 hours of treatment, spheroids were subjected to a Brite-Glo assay (Promega) according to the manufacturers’ instructions.

PE-conjugated anti-mouse CD206 (Clone M1), PE-conjugated anti-mouse MHCII, PE-conjugated anti-mouse PDL1, PerCP-conjugated anti-mouse CD40, PE-Cy7-conjugated anti-mouse CD86, PE-conjugated anti-mouse CD80 and corresponding isotype controls were purchased from BD Pharmingen (San Diego, CA). Penicillin and streptomycin, DCFH-DA probe, HRP-conjugated goat anti-mouse IgG (H + L) are from Beytotime (Haimen, Jiangsu, China). Annexin V/7-AAD apoptosis kit was purchased from BD Pharmingen (San Diego, CA). Alexa Fluor® 647 AffiniPure F(ab′)₂ Fragment Rabbit Anti-Mouse IgG (H + L) was purchased from Jackson Immunoresearch (West Grove, PA, USA). Matrigel was purchased from Sigma (St. Louis, MO).