By surface labeling and confocal immunofluorescence, KCNE2 appeared more abundant at the cell surface compared to KCNE1, which exhibited greater co-localization with the ER-marker calnexin.
The suspension of released cells was collected by centrifugation, washed and surface labelled with the appropriate fluorochrome-conjugated monoclonal antibodies (mAbs): CD45-PerCP, CD3-FITC, TcRγ δ-PE and CD103-PE (BD Biosciences, San Diego, CA).
After incubation, the cells were surface labeled with anti-CD8 Ab as described earlier, followed by intracellular staining for IFN-γ, granzyme-B, and TNF-α.
Animal cap cells were surface labeled with cleavable biotin, induced with activin, and plated on FN.
In the case of cell surface versus intracellular labelling, cells were first cell surface labelled with mAb CD133/1 in the cold prior fixation, as described (Corbeil et al, 1999), followed by the saturation of remaining mouse epitopes with unconjugated AffiniPure rabbit-α-mouse IgG (H + L) Fab fragment.
The cell-surface antigens recognized by these antisera were examined using cell-surface labelling with I, followed by immunoprecipitation of soluble extracts of the cells and polyacrylamide-gel electrophoresis of the immunoprecipitates in the presence of sodium dodecyl sulphate (SDS).
In all fluorescent activated cell sorter (FACS) experiments, cells were pre-coated with anti mouse CD16/CD32 (BD Pharmingen,) to block unspecific binding, and T cells were identified by cell-surface labeling with APC-labeled anti-Thy1.2 (BD Pharmingen).
To visualize the acquisition of exogenous OVA peptide by internalized H-2Kb molecules, DCs surface-labeled with H-2Kb-FITC antibody as described above were incubated at 37°C in complete RPMI containing either 5 mg/mL ovalbumin protein (Worthington, NJ) or bovine serum albumin control protein.
Early studies used metabolic labeling and surface labeling techniques with SDS-PAGE to detect altered protein patterns [ 1- 3].
Apoptosis was measured with the Annexin V surface labelling, DNA fragmentation was assessed by the TUNEL assay (both using assay kits from Guava Technologies, Hayward, CA, USA).
