Anti human cxcr4 monoclonal antibody

Indirect fluorescent immunocytochemistry was performed for CXCR4, MUC5AC, and Smad2/3 on cells grown in 4-well chamber slides. Cells were fixed in 4% paraformaldehyde at room temperature for 20 min. After three times washing in PBS(-), they were incubated with anti-human CXCR4 monoclonal antibody (R&D Systems, Minneapolis, MN), anti-human MUC5AC antibody (Santa Cruz Biochemistry, Santa Cruz, CA), or anti-human Smad2/3 antibody (BD Biosciences, Bedford, MA) followed by incubation with a 1:1000 dilution of donkey anti-mouse Alexa 488-conjugated serum (Invitrogen, Grand Island, NY) for 1 hr at room temperature. Cell nuclei were stained with 4’, 6-diamidino-2-phenylindole dihydrochloride (DAPI) (Invitrogen, Carlsbad, CA). For cell-lineage assay, sorted single CXCR4⁺ small cells were stained by CellTracker Green (Invitrogen) for tracing the viable cells.

Recombinant CXCL12 (catalogue number 350-NS), CCL19 (catalogue number 361-MI-025), CCL21 (catalogue number 457-6C-025) and anti-human CXCR4 monoclonal antibody (catalogue number MAB173) were purchased from R&D Systems (Abingdon, OX14 3NB, UK). Alexa Fluor 488 anti-human CCR7 antibody (catalogue number 353206) and the recommended isotype control (catalogue number 400233) were purchased from Biolegend (London, NW5 1LB, UK). Ultrapure^TM low-melting agarose (catalogue number 16520–050) was purchased from Invitrogen (Paisley, PA4 9RF, UK). CXCR4 antagonist AMD3100^{65 (link)} (catalogue number 3299), was purchased from Tocris Biosciences (Missouri 63021, USA). CCR7 Antagonist ICT13069 was synthesised at the Institute of Cancer Therapeutics (Bradford, BD7 1DP, UK). Cascade Blue-10 kDa (Catalogue number D1976) and Fluorescein-40 kDa (Catalogue number D1845) dextrans were purchased from ThermoFisher (Loughborough, LE11 5RG, UK). Vectashield hardset antifade mounting medium with DAPI (catalogue number H-1500) was purchased from Vector laboratories, (Peterborough, PE2 6XS, UK). Bovine serum albumin (catalogue number A2153) was purchased from Sigma-Aldrich (Gillingham, SP8 4XT, UK).