Dithiothreitol dtt

Cells or tissues were homogenized in RNA Stat60^® and total RNA extracted using standard phenol-chloroform protocols followed by DNase treatment of the extracted RNA using RNA-II purification kit (Nachery-Nagel). A total of 100 ng of RNA per sample was converted into cDNA using Superscript II (Life Technologies) at 42 °C for 50 min, 70 °C 15 min, in the presence of 5 uM oligo (dT)_16–18, 5 mM Dithiothreitol (DTT), 0.5 mM dNTPs (all Life Technologies), 8 U RNAsin (Promega), 50 mM Tris-HCl pH 8.3, 75 mM KCl and 3 mM MgCl₂. The cDNA was treated with 2.5 U RNase H (Affymetrix) at 37 °C for 20 min to remove any remaining RNA residues.

All reagents were purchased from Sigma-Aldrich (UK) or Life Technologies (UK) unless otherwise stated. Aliquots of Dithiothreitol (DTT) (Life Technologies) were pre-prepared in distilled H₂O and stored at −20^°C. The wash solution (mucolytic solution) used in the initial steps of enterocyte isolation contained 1% penicillin/streptomycin and an aliquot of DTT dissolved in Hanks Buffered Saline Solution (HBSS) with MgCl₂ and CaCl₂ which gave a final concentration of 1 mM. Fresh wash solutions were made for each isolation due to DTT's reduced activity at room temperature as previously observed by Goodyear et al. (2014) (link). Solution formulations can vary between manufacturers and significantly impact the type and success of primary cultures of RT tissue as previously observed by Ganassin and Bols (1996) (link) in cultures of RT spleen. As such, trypsin (25050014), versene (15040033) and trypsin/EDTA (25300054) were purchased from Life Technologies. The enzyme digestion solution contained 0.1% collagenase D (COLLD-RO, Roche) and dispase II (D4694, Sigma-Aldrich) (equivalent to 1 mg/ml) with DNase (0.1 mg/ml) and 1% FBS/BSA. This solution is pre-warmed to 37°C prior to enterocyte isolation as is routine in many primary culture protocols.