Pe conjugated antibodies

The immunophenotype of purified (un)stimulated NK cells was evaluated using PE-conjugated antibodies (BD Biosciences) and flow cytometry for the following surface markers that might have a stimulating or inhibiting effect on NK cell functions: NKG2D, DNAM-1, PD-1, TIM-3, LAG-3, NKp46, FasL, TRAIL and 2B4. In addition, corresponding ligands on the PCC and (primary) PSC were measured using PE-conjugated antibodies and flow cytometry for the following markers: MICA/B, ULBP1, ULBP2,5,6, ULBP3, ULBP4 (NKG2D ligands; antibodies all from R&D Systems), Galectin-9 (TIM-3 ligand; antibody from BD Biosciences), PD-L1 and PD-L2 (PD-1 ligands; antibodies both from BD Biosciences). Corresponding isotype controls were included for all samples and served as negative controls. All samples were measured on a FACSCan flow cytometer (BD Biosciences).

The expression of TRAIL receptors on surface membrane was assessed by flow cytometry using PE-conjugated antibodies (R&D Systems) as previously described [14 (link), 18 (link)]. Briefly, cells were grown at 70% - 80% confluence and harvested by incubation with Trypsin (0.25% w/vol)-EDTA (0.53 mM) at 37 °C for 3 min. Trypsinization prevents the formation of cell clumps after harvesting which facilitates the subsequent flow cytometry analysis. The resulting cells were immediately washed twice in PBS. Aliquots (1 X 10⁵cells) were incubated in 25 μL PBS containing 1% goat serum for 15 min at room temperature. Afterwards, cells were incubated with 10 μg/mL anti-DR4-PE or anti-DR5-PE (mouse IgG1-PE and IgG2b-PE as respective control) for 45 min at 4°C in the dark. Duplicate samples were incubated with the respective control IgG-PE (mouse IgG1-PE and IgG2b-PE) under the same conditions. Cells were then washed twice with PBS and resuspended in 0.5 mL PBS for final analysis. All cell lines were processed under the same conditions which ensure a direct comparison of the relative expression levels of DR4 and DR5 on surface membrane.

Parental and resistant cells were collected by trypsinization, washed in 1x PBS and counted. Single-cell suspensions were incubated in a 5% BSA solution containing anti-AXL or TYRO3, PE-conjugated antibodies at 1:50 (R & D Biosystems) for 40mins in the dark at room temperature. Cells were passed through a cell strainer to collect single cells and were protected from light until they were quantified using a Beckman-Coulter Cytomics FC500 flow cytometer with at least 10,000 events counted per test. Histograms were used to compare intensity of staining between unstained, parental and resistant cell line samples. Median fluorescence intensity was calculated for each sample and t-tests were used to quantify differences.