Snarf 1 dextran

All reagents used for the preparation of polyacrylamide hydrogels with pH-sensitive fluorescent microcapsules and further procedures were of analytical purity and were used without additional purification. Acrylamide (#A1089) was purchased from AppliChem GbmH (Darmstadt, Germany). The following reagents were purchased from Helicon (Moscow, Russia): N,N′-methylenebisacrylamide (#22797959), ammonium persulfate (APS, #H-248614), N,N,N′,N′-tetramethylethane-1,2-diamin (TEMED, #68604730). The conjugate of seminaphtharhodafluor-1 with dextran (SNARF-1-dextran; #D-3304) was bought from Thermo Fisher Scientific (Eugene, OR, USA). Poly (allylamine hydrochloride) (PAH; #283215) and poly (sodium 4-styrenesulfonate) (PSS; #243051) were provided by Sigma-Aldrich (produced in St. Loius, MO, USA and Overijse, Belgium, respectively).

For the labeling of OVA with pHrodo, a stock solution of 9 mM pHrodo Green STP ester (Thermo Fisher) in DMSO was mixed with 10 volumes of 10 mg/ml OVA and incubated for 2 h at RT. DCs were pulsed with 1 mM pHrodo-conjugated OVA and 1 μg/ml LPS in the presence or absence of 500 μM α-tocopherol for 15 minutes, followed by a chase of 150 minutes. The fluorescence intensities of pHrodo was measured by flow cytometry at 20, 40, 60, 90, 120 and 150 minutes after starting with the chase (excitation: 488 nm; emission: 530/30 nm). pH was calibrated by measuring the fluorescence of pHrodo in a standard solution series of known pH.
For the SNARF-1 experiments, DCs adhered to glass bottom microdishes were washed and incubated for 30 minutes with 1 mg/ml SNARF-1 dextran 10,000 MW (D3303, Thermo Fisher) with or without 500 μM α-tocopherol or 1 μM PAO. DCs were imaged with a Leica SP8 confocal microscope and a 63 × 1.20 NA water immersion objective (540 nm excitation; 550–600 nm green and 620–700 nm red emission peaks of SNARF-1). The pH calibration curve was created by imaging SNARF-1 dextran-containing DCs that were incubated with buffers of different pH (10 mM phosphate buffer, 150 mM NaCl) and 0.003% Triton-X100.