Synergy h1 multi mode microplate reader

Tight junction integrity and paracellular permeability was measured using size-selective fluorescently labeled dextran, as previously described^{70 (link)}. Human AECs transfected with shRNA to silence CDH26A or scramble control were taken to ALI. After reading transepithelial resistance, media was replaced with 5% FCS 250 μL on the apical membrane and 1 mL in the basal chamber and placed back into the incubator to equilibrate. An empty filter with no cells was used as a control for flux and 25 μL of rhodamine B-isothiocynate-dextran (RbITC) 70,000 mw and 25 μL of fluorescein isothiocynate-dextran (FITC) 4000 mw were added to the apical chamber for a final concentration of 2 mg/mL for each tracer. After 4 h, 100 μL was collected from the basal chamber to measure transit of tracer from apical to basal chamber and read (FITC ex 485 nm/em 544 nm, RbITC ex 520 nm/em 590 nm) in a black 96 well microplate on a Biotek Synergy H1 Multi-mode Microplate Reader.

Total RNA was isolated from approximately 10 million LCLs (both LCL#1 and LCL#89) either left untreated (DMSO control) or treated with 1 μM MG132 for 12 h using TRIzol reagent according to the manufacturer’s instructions (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA), followed by cDNA preparation using iScript cDNA synthesis kit (BIO-RAD, Hercules, CA, USA) as per manufacturer’s protocol. RNA and cDNA quality and quantity were checked using Synergy H1 Multimode Microplate Reader (BioTek Instruments, Inc., VT, USA). qPCR analysis was performed using iTaq Universal SYBR Green Supermix (BIO-RAD, Hercules, CA, USA) in CFX Connect Real-Time PCR detection System (BIO-RAD, Hercules, CA, USA) with the following thermal profile– 1 cycle: 95°C for 10 min; 40 cycles: 95°C for 15 sec followed by 60°C for 1 min; and finally the dissociation curve at– 95°C for 1 min, 55°C 30 min, and 95°C for 30 sec. Unless and otherwise stated, each sample was performed in duplicate and calculation was made using a 2^−ΔΔCT method to quantify relative expression compared with housekeeping gene controls–B2M, GAPDH and RPLPO. The real time PCR primers used in this study were designed from Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and listed in S2 Table. Real-time PCR primers were obtained from Integrated DNA Technologies, Inc. (Coralville, IA, USA).