Phenol red free dmem

The hCRY2-LUC fusion protein expressing vector was created by inserting a CRY2-Luc cDNA between EcoRI and BamHI sites in the p3×FLAG-CMV-10 vector. HEK293 cells were transfected with 50 ng hCRY2-LUC vectors and cultured for 24 hr. The culture medium was replaced with recording medium [phenol-red free DMEM (Sigma Aldrich) supplemented with 10% fetal bovine serum, 3.5 mg/ml glucose, 50 U/ml penicillin-streptomycin (Thermo Fisher Scientific), 0.05 mM luciferin, and 10 mM HEPES-NaOH; pH 7.0] containing 100 μg/ml cycloheximide (CHX; Santa Cruz Biotechnology Inc., Santa Cruz, CA). Luciferase activity of hCRY2-LUC was recorded at 10-min intervals at 37°C with a LumiCycle 32 instrument (Actimetrics). The luminescence signals were fitted to an exponential function to quantify the half-life of CRY2-LUC. KL001 (Cayman Chemical, Ann Arbor, MI) was diluted in DMSO to a final concentration of 20 mM.

RN cultures were seeded at 4000 cells/well in a 96 well plate and permitted to settle for 24 h before washing well before labelling with 0.5 μM of the fluorescent probe di-8-ANEPPs (Invitrogen, from 2 mM stock solution in ethanol) for 1.5 h in phenol-red free DMEM (Sigma-Aldrich) (Davis et al., 2015 ). After this time the ratiometric di-8-ANEPS fluorescence intensity at excitation of 420/520 nm and emission of 670 nm using a Safire plate reader for each cell population was recorded before and 10 min after cells were treated with varying concentrations of TPGS for 10 min. The change in fluorescence ratio of di-8-ANEPPs indicates a change in the membrane dipole potential on addition of an agent of interest. The dissociation constant (K_d) of the interaction of TPGS for neuronal cells was determined by fitting the change in di-8-ANEPPs fluorescence ratio to a hyperbolic binding equation as described previously (Davis et al., 2010 (link)).

Cell suspension in phenol red-free DMEM (Sigma, USA) supplemented with 10% FBS was plated in 96 well flat-bottom tissue culture plates (BD) at a pre-determined concentration of 10⁴ cell/well in a final volume of 100 μl. The optimal cell density and incubation time were determined for each cell line by titration and kinetic experiments where optimal conditions yielded the optical densities (OD) between 0.3-0.8 in the logarithmic portion of the standard curve. After that, 100 μl of phenol red-free DMEM containing LPS or LTA (final concentrations: 1–1000 ng/ml) was added to the test wells in triplicate, while control wells received the same volume of vehicle. After 48 hours, XTT was prepared at 1 mg/ml in pre-warmed (37°C) serum and phenol red-free DMEM. PMS was prepared at 5 mM (1.53 mg/ml) in PBS. Fresh XTT and PMS were mixed together at the appropriate concentrations. For a 0.025 mM PMS-XTT solution, 25 μl of the stock 5 mM PMS was added per 5 ml of XTT (1 mg/ml). 50 μl of this mixture (final concentration, 50 μg of XTT and 0.38 μg of PMS per well) was added to each well and the plates were incubated at 37°C, 5% CO2 for 4 hours. Thereafter, the OD values were read at 450 nm. For each cell line, the mean OD value of wells without cells was subtracted from that of test and control wells to calculate net OD value of each well.

Immortalized mouse brain endothelial cells, bEnd.3, were obtained from American Type Culture Collection (Manassas, VA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT), 3 mM L-glutamine (Sigma, St. Louis, MO) and 1% penicillin-streptomycin (Sigma, St. Louis, MO). bEnd.3 s were plated at 6 × 10⁴ cells/cm² in Transwell filters (1.1 or 4.67 cm² membrane area, 0.4 μm pores; Corning, Lowell, MA) coated with fibronectin (Sigma, St. Louis, MO) and cultured for 4–5 days before transport experiments. Experimental media consisted of phenol-red free DMEM (Sigma, St. Louis, MO) supplemented with 1% bovine serum albumin.

To measure the cellular ROS levels, foreskin fibroblasts were washed twice with PBS and treated with 10 μM of DCF-DA in serum-free DMEM in the dark at 37 °C and 5% CO₂ in an incubator for 30 min. The cells were then washed twice with PBS and once with phenol-red-free DMEM (Sigma-Aldrich), and they were analyzed using a fluorescence microscope. The fluorescence intensity was measured using ImageJ software. To measure the cell viability, fibroblasts were fixed with 3.7% paraformaldehyde in PBS and stained with 0.005% crystal violet. The stained cells were lysed with 1% SDS, and the absorbance was measured at 600 nm [29 (link)].

Human embryonic kidney (HEK) 293T and baby hamster kidney (BHK)-21 cells (Central Services Unit, The Pirbright Institute, UK) were used for pseudoparticle generation and mVNTs and were maintained using DMEM-10%: Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich) supplemented with 10 % foetal bovine serum (FBS; Life Science Production), 1 % sodium pyruvate, NaP solution (Sigma-Aldrich) and 1 % penicillin/streptomycin (Pen-Strep; 10 000 U ml⁻¹; Life Technologies Ltd). HEK293T cells (Cell Servicing Unit, The Pirbright Institute, UK) stably expressing Lenti-rLuc-GFP 1–7, or separately, Lenti-rLuc-GFP 8–11 were used for all fusion assays and mFITs and were maintained using PRF-DMEM-10%: phenol red-free DMEM (Sigma-Aldrich) supplemented with 10 % FBS (Life Science Production), 1 % NaP (Sigma-Aldrich), 1 % Pen-Strep (10 000 U ml⁻¹; Life Technologies Ltd) and 1 % l-glutamine 200 mM (Sigma-Aldrich).

GimRET-fused Nups or importin β were co-expressed with mPlum (Clontech)-fused histone H3 in HeLa cells cultured in DMEM (SIGMA) supplemented with 10% FBS. Before the microscopic observations, the culture medium was replaced with phenol red-free DMEM (SIGMA) supplemented with 10% FBS and 8 μM l-glutamine. The stage-top chamber (Tokai-hit) was filled with moisture using distilled water and 5% CO₂, and was maintained at 37 °C. Images were captured every 2 min until the cell entered the early G1 phase. Signal intensity at chromosome rim was measured manually using MetaMorph (Molecular Devices) software.