Apoptosis levels were demonstrated by Annexin-V staining and propidium iodide (PI) exclusion using the FITC Annexin-V Apoptosis Detection Kit I (BD Pharmingen, San Diego, CA, USA).
Cell viability was assessed at initiation and harvest of cultures and determined to be 87 100% using Trypan blue (Merck, Darmstadt, Germany) exclusion and Cytotoxicity Detection Kit (LDH) (no. 11644793001, Roche, Mannheim, Germany) according to the manufacturer's instructions.
Purification was done using i) phase separation with TRIzol and ii) column exclusion with the RNeasy Mini Kit (Qiagen, Germany) according to the manufacturers instructions.
After 24 h incubation with C7a, the cytotoxic activity was determined by measuring cell viability by two different methods, Trypan Blue exclusion and the Cell Proliferation kit (MTT, Roche Diagnostics Comp., Indianopolis, IN), following the manufacturer's instructions.
Survival rates of DMSO-treated cells were determined by Invitrogen live/dead viability/cytotoxicity kit and trypan blue exclusion assay.
Differences among platforms would presumably be based on variations in depth of coverage and coverage efficiency rather than due to the selective exclusion of some regions by specific capture kits (for example, the exclusion of UTRs by SSCR, NX, NG).
Enriched poly(A)+ RNA was ligated to the 5′-RACE adapter provided in the FirstChoice kit without treatment with CIAP and TAP for exclusion of all 5′-capped mRNA.
In terms of specific challenges the team has encountered filming in the radioactive Chernobyl exclusion zone, Tereshchenko says they had to be kitted out with personal dosimeters and, in the high radiation level locations, were also donning protective clothes and shoes and using special housing to protect their kit.
Cell viability was assessed by trypan blue exclusion or by MTT cell proliferation assay using CellTiter 96 kit from Promega (Madison, WI, USA).
Cell proliferation was determined by using a WST-1 kit (Roche, Indianapolis, IN) or by trypan blue exclusion method as described previously [27].
