PMC:7108609 / 28990-31638
Annnotations
2_test
{"project":"2_test","denotations":[{"id":"18818423-2460087-45167076","span":{"begin":394,"end":398},"obj":"2460087"},{"id":"18818423-8737251-45167077","span":{"begin":417,"end":421},"obj":"8737251"},{"id":"T88030","span":{"begin":394,"end":398},"obj":"2460087"},{"id":"T50163","span":{"begin":417,"end":421},"obj":"8737251"}],"text":"Flow cytometry, immunofluorescence, and Western blot analyses\nCells at near confluence were detached by a brief 0.025% trypsin/0.02% EDTA treatment. Viable cells, 2 × 105 per well of 96 culture microtiter plates, were incubated with primary anti-H type 2 or broad reactive anti-A monoclonal antibodies 19-0LE and 3-3A respectively, in PBS containing 0.1% gelatin for 30 min at 4°C (Bara et al. 1988; Mollicone et al. 1996). After three washes with this same buffer, a 30-min incubation with the secondary anti-mouse IgG FITC-labeled antibody (Sigma) was performed at 4°C. After washing, fluorescence analysis was performed on a FACSCalibur (Becton-Dikinson, Heidelberg, Germany). The expression of the EGFP-S protein construct was detected by its autofluorescence on the FL1 channel.\nCHO cells transfectants, cultivated on glass lamellae, were fixed by the addition of 2% formaldehyde in a culture medium for 10 min. After washing in PBS, the cell monolayer was incubated with the anti-A mAb 3-3A at 0.5 μg/mL in PBS for 1 h and washed thrice in PBS before incubation for 30 min with TRITC-labeled anti-mouse IgG (Sigma) diluted at 1/400. After three final washings in PBS, slides were mounted in Mowiol and observed under a Leica TCS SP (Heidelberg, Germany) confocal fluorescence microscope. Negative controls were incubated with the secondary antibody alone.\nConfluent cells (CHO Fut2 simple transfectants, CHO Fut2/A double transfectants, and CHO Fut2/A/PS triple transfectants) were rinsed with ice-cold PBS, pH 7.2, and then recovered by scraping. After washing with ice-cold PBS, cells were solubilized in 50 mM potassium phosphate, pH 6.0, containing 2% (v/v) triton X-100 on ice for 30 min. Following a centrifugation at 13,000 × g for 10 min, the supernatant was collected and the protein concentration was determined using the BC assay protein quantification kit (Uptima, Montluçon, France). Thirty micrograms of total proteins of each extract were separated on 8% SDS–PAGE under reducing conditions and electrotransferred to immobilon P sheets (Millipore, Bedford, MA). Immunoblots were saturated for 1 h at room temperature with Western blocking reagent (Roche Diagnostics GmBH, Mannheim, Germany) and strips were cut and incubated overnight at 4°C with the anti-A 3-3A mAb at 2 μg/mL in antibody Western blocking reagent. Following three washes for 15 min with TBS containing 0.05% Tween 20, strips were incubated with horseradish peroxidase-labeled anti-mouse IgG (H + L) (Beckman Coulter, Fullerton, CA) for 1 h at room temperature. After three final washes, detection was performed with a chemiluminescence kit (Roche Diagnostics)."}