Dtx 800 multimode plate reader

GSH/GSSG-Glo assay kit (Promega, Cat.V6611) was used for glutathione measurements. After 48h 800μM H₂O or BSO pretreatment, 10,000 H₂O-treated HaCaTs cells/per well or 15,000 BSO-treated cells/per well were transferred into white 96 well plates. Then, another 24h H₂O or 800μM BSO treatment was followed. To measure total GSH levels, 50μl per well total glutathione lysis reagent (containing Luciferin-NT, passive lysis buffer) was added to the cells. To measure oxidized GSSG, 50ul per well oxidized lysis reagent (containing Luciferin-NT, passive lysis buffer, and 25mM NEM) was added. Plates were incubated at room temperature and shaken for 5 minutes, then 50μl per well luciferin generation reagent (containing 100mM DTT, glutathione S-transferase and GSH reaction buffer) was added. The plate was shaken and incubated at room temperature for another 30 minutes. Finally, 100μl/well luciferin detection reagent was added and the plate was shaken and incubated at room temperature for another 15 minutes. The luminescence was measured using a DTX-800 multimode plate reader (Beckman Coulter). Standard curves were generated using serial dilutions of standard GSH (provided by the kit) ranging from 16μM to 0.25μM.

HaCaT GFP-BAP cells were plated at 50,000 cells per well in a 24-well plate. Cells were infected the following day with L2-BirA virus at 2 x 10⁸ viral genomes/well. At 24 hours post-infection (p.i.), the cells were washed once with PBS and lysed in 100 μl reporter lysis buffer (Promega E3971). Luciferase activity was measured on a DTX-800 multimode plate reader (Beckman Coulter) using luciferase assay reagent (Promega E4550) according to the manufacturer’s instructions. A fraction of these lysates were blotted for GAPDH to normalize luciferase activity.

L-Buthionine-(S, R)-sulfoximine (BSO, Santa Cruz, CAS 83730-53-4) was prepared at 200mM in water. Cells were pretreated with 800μM BSO for 72h prior to experiments. 50,000 cells/well of water or BSO-treated HaCaTs were seeded in 24 well plate the day before infection. Cells were infected with HPV16 at 2x10⁸ viral genome equivalents (pGL3 copies) per well or 10,000 luciferase expressing HAdV-5 vector particles per cell. At 24h post-infection, cells were lysed in 100μl RLB. 100μl luciferase assay reagent (Promega E1483) was added into 20μl cell lysate and luciferase activity was measured using a DTX800 Multimode plate reader (Beckman Coulter). The remainder of the cell lysate was collected for western blots and GAPDH immunostaining. GAPDH bands were quantified by densitometry using ImageJ software [41 (link)] to normalize the luciferase data.