Ab18407

Cells were cultured in 8-well chamber slides and irradiated by LINAC at 5, 15 and 25 Gy doses. Cells were fixed 24, 72 or 120 h after radiation with 4% paraformaldehyde prior to blocking and incubation with anti-ICAM-1 antibody (ab119871, Abcam) or IgG2b isotype (ab18541, Abcam) that was fluorescently tagged using Mix-n-Stain^TMCF^TM555 antibody labelling kit (Sigma-Aldrich), or anti-VCAM-1 antibody (ab61993, Abcam) or IgG1 isotype (ab18407, Abcam) labelled using Mix-n-Stain^TMCF^TM640 antibody labelling kit (Sigma-Aldrich). Nuclei were stained using 4',6-diamidino-2-phenylindole (DAPI) (1 μg/mL, Life Technologies). Images were captured with fixed parameters using a confocal microscope (SP5, Leica Microsystems, Wetzlar, Germany) and analyzed with ImageJ.

The liquid-overlay technique was used for spheroid formation (Carlsson and Yuhas, 1984 (link)). Briefly, 96-well culture dishes were coated with 50 µl 1% low-melt agarose to form concave non-adhesive wells. Per well, 600 cells were seeded in 50 µl assay medium containing 0.5% penicillin/streptomycin. Drugs were diluted to their respective dilution and 50 µl drug solution was added to each well. Cytochalasin D (Enzo), nocodazole (Sigma-Aldrich), and PF-573228 (Sigma-Aldrich) were dissolved in DMSO (Sigma-Aldrich) with a final concentration of 0.5%. For all drugs, the appropriate, non-toxic concentration for each cell line was determined beforehand.
To inhibit E-cadherin, the DECMA-1 antibody (ab11512, Abcam) or the control IgG1 antibody (ab18407, Abcam) were used at 10 µg/ml. Well plates were centrifuged at 400 g for 4 min and then subjected to further analyses.