Mal 2 lectin

MAL-II expression was analysed using the MAL-II lectin (Vector Laboratories, B-1305-2) conjugated to streptavidin AlexaFluor 647 (Abcam). 1x10⁵ cells were pelleted by centrifugation, washed in PBS-T, resuspended in 1X carbo-free blocking solution (Vector Laboratories) and labelled with 2 μg/mL MAL-II lectin for 30 min at 4 °C. Cells were washed and stained with streptavidin Alexa Fluor 647 (Abcam) for 15 min at 4 °C, before repeating washes and resuspending in PBS. Propidium iodine was used to discriminate between live and dead cells and cells were processed through a FACSymphony flow cytometer. Data was analysed and histograms generated using Red Matter App (v2.01) and plotted using a log scale.

Human pancreatic cancer cells were dissociated with trypsin, washed twice with HBSS containing 2% fetal bovine serum and 10 mM HEPES (staining buffer). Cells were stained and incubated on ice for 30 minutes with the following primary antibodies: ABCG2 (R&D Systems Inc, 1:50), FITC-conjugated CD24 (BD Biosciences, 1:100), APC-conjugated CD44 (BD Biosciences, 1:100), biotinylated SNA lectin (Vector Laboratories, 1:100), or MALII lectin (Vector Laboratories, 1:100). After stained with appropriated secondary antibody, the stained cells were analyzed by FACSCalibur (BD Biosciences).