Glo lysis buffer

The human embryonic kidney (HEK293T) cell line was cultured at 37 °C under 5% CO₂ in Eagle’s Minimum Essential Medium (EMEM) containing 10% heat-inactivated fetal bovine serum (HI-FBS) and 1% Penicillin/Streptomycin medium. HEK293T cells were seeded at 1 × 10⁵ cells/well in 12-well plates and cultured overnight at 37 °C under 5% CO₂ with 1 mL/well full growth media for 24 hours. Then the full growth media was replaced with 400 μL Opti-MEM containing polyplex of 1.5 μg DNA in each well. After 4 hours incubation, the transfection media was replaced with full growth media, and the cells were incubated for 24 hours and 36 hours for pGFP and pLuc expression analysis, respectively.
For the pGFP transfection study, the transfected cells were studied by fluorescence microscopy and flow cytometry. The apoptosis assay was carried out through flow cytometry after Annexin-V (AV) and Propidium Iodide (PI) staining.^{64 (link)} For the pLuc transfection study, the transfected cells were lysed in 400 μL of 1X Glo Lysis Buffer (E2661, Promega). Then the lysate was transferred to a 96-well plate and mixed with an equal amount of Steady-Glo Assay Reagent (E2510, Promega). The luminescence intensity was measured through the Fluoroskan Ascent FL after 20 minutes.

Supernatant was removed from transfected cell monolayers, and cells were briefly washed with PBS and lysed using 100 μl 1x Glo Lysis Buffer (Promega®) per well in a 12-well plate. The oxidation reaction was catalyzed by the addition of 10 μl cell lysate to 10 μl room temperature Bright-Glo Luciferase Assay System (Promega®) substrate. Luciferase activity was measured using a luminometer with values normalized to protein content of the extract using a protocol as previously described (Arnold et al., 2006 (link)).

BNL-CL2 (BALB/c embryonic normal liver) cells were cultured until passage five in standard DMEM (4.5g/L glucose, L-glutamine and sodium pyruvate) supplemented with 10% FBS and 1% PenStrep (Thermo) at 37 °C, 5% CO₂. Approximately 2 × 10⁵ cells per well were plated in a tissue culture treated 24-well dish (Becton-Dickinson). After 24 hours, media was replaced with DMEM without glucose, L-glutamine and sodium pyruvate supplemented with 1% FBS for 4 hours prior to transfection. LXRα signaling plasmids and control plasmids (Cignal Reporter Assay Kit, Qiagen) were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Transfection of each plasmid was performed in triplicate in two independent experiments. Two hours post transfection, media was replaced with DMEM supplemented with 1% FBS and 1% PenStrep with the addition of PANDER (0.25 nM, 0.5 nM and 1nM) for 18 hours. Cells were washed with PBS and lysed in 100 μl of 1X Glo Lysis Buffer (Promega), utilizing the Dual-Luciferase Reporter Assay System (Promega) for luciferase activation. Signal emission was detected with a Monolight 3010 luminometer (Analytical Luminescence Laboratory, San Diego, CA).

Whole cell extracts from cultured cell lines were prepared using 1x GLO lysis buffer (Promega, E266A). One third volume of 4x Laemli buffer was added to one volume whole cell extract samples, then incubated at 95°C for 5 minutes, and sonicated for 15 minutes at 4°C (amplitude 100; pulse interval 15 seconds on, 15 seconds off). Approximately 30 ug of protein were separated by SDS-PAGE (BioRad, 1610175), transferred to PVDF membrane (BioRad, 1620177), blocked according to antibody manufacturers specification, and incubated overnight in appropriate primary antibody then incubated in IRDye or peroxidase conjugated goat anti-mouse or anti-rabbit secondary antibodies for 1 hour at room temperature. Protein was then detected using ECL reagent (BioRad, 1705061) or the Licor Odyssey imaging system.