Fig 1: Overshoot of CXCR6 and CD271 in human melanoma sorted cells.CXCR6- or CD271- negative cells are sorted from IgR39 cells as described in Methods and plated under standard growth conditions. Cells are collected 3, 10 and 20 days after sorting, and analyzed by flow cytometry. Non-specific mouse IgG is used as isotype control (Isotype). The distribution of the markers in unsorted cells is also reported (U). Flow cytometry is performed using a FACSAria flow cytometer (Becton, Dickinson and Company, BD, Mountain View, CA). Data are analyzed using FlowJo software (Tree Star, Inc., San Carlos, CA). For each flow cytometry evaluation, a minimum of 5 × 105 cells are stained and at least 50000 events are collected and analyzed. (a) The level of expression of each markers in unsorted cells (U) and at different times after sorting is reported as flow cytometric analysis of a minimum of 5 independent experiments. (b) Unsorted cells (U), CXCR6-positive, and CXCR6-negative cells after 10 days sorting are fixed with 3.7% paraformaldeide and incubated with polyclonal anti-CXCR6 antibody (1:400, Abcam, Ab8023) overnight. Then the cells are incubated with the secondary antibody (anti rabbit Alexa488 1:250) for 1 h and the nuclei counterstained with DAPI. The slides are mounted with Pro-long anti fade reagent (Invitrogen) and the images acquired with a Leika TCS NT confocal microscope.
Supplier Page from Abcam for Anti-CXCR6 antibody