Mouse igg2a fitc

Vacutainers for blood collection K2-EDTA (Cat: 367863) and serum separation (Cat: 367812) were purchased from Becton Dickinson Biosciences. Fluorescent antibodies such as anti-human CD133-Phycoerythrin (PE) (Cat: 130-080-801) were purchased from Miltenyi Biotech Anti-human CD34-Fluorescein isothiocyanate (FITC) (Cat: 343604), anti-human VEGFR2-Allophycocyanin (APC) (Cat: 359916) and isotype control antibodies like mouse IgG2a-FITC (Cat: 400207) and mouse IgG1k-PE (Cat: 400113), were purchased from BioLegend. Other dry chemicals were purchased from Sigma-Aldrich.

To label cell surface immunoglobulins, cells were washed in PBS and resuspended in PBS/5% fetal calf serum containing fluorochrome-labelled anti-Ig antibodies. After 1 h incubation, cells were washed in PBS, fixed in 2% paraformaldehyde for 20 min, washed again and assessed by FACScalibur analyser (BD Biosciences, Oxford, UK). Isotype and fluorochrome-matched non-targeting antibodies, added at equal concentrations, were used to set the background fluorescence. The following BioLegend (BioLegend UK, London, UK) antibodies were used in this study: PE anti-human Ig light chain λ (316607), PE Mouse IgG2a, κ isotype Ctrl (FC) (400213), PE anti-human Ig light chain κ antibody (316507), PE Mouse IgG1, κ isotype Ctrl (FC) antibody (400113), FITC anti-human IgD antibody (348205), FITC Mouse IgG2a, κ isotype Ctrl (FC) antibody (400209). FITC Fab2 anti-human IgG (F0185), RPE Fab2 anti-human IgM (R5111), and corresponding isotype controls (control reagent, Rabbit F(ab')2/FITC, X0929, and control reagent, Rabbit F(ab')2/RPE, X0930) were obtained from Dako (Agilent Technologies, Cheadle, UK).