Cells

Example 5

MTT cytotoxicity assays were used to assess the cytotoxicity of RGQDs/Nd-GQDs/Tm-GQDs. HeLa cells were plated in a 96-well plate with 5000 cells per well (100 μL/well) and kept in an incubator overnight at 37.1° C. while maintaining the CO₂/air ratio of 1:19. After 24 h of incubation, the samples were added into each well at different concentrations for different materials (0 to 70 μg/mL, 1 mg/ml, 0.25 mg/ml for RGQDs, Nd-GQDs, Tm-GQDs, respectively). After 24 h of incubation, the medium was replaced by 100 μL of 1 mg/mL thiazolyl blue tetrazolium bromide. After 4 h of further incubation, MTT (3-(4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was replaced with 100 μL of DMSO (dimethyl sulfoxide) to solubilize the precipitation. Reduction in MTT influences the metabolic activity of living cells, which can be assessed with absorbance measurements because living cells metabolize the MTT and form a highly absorbing purple colored byproduct known as formazan. The absorbance (essentially the cell viability) of the final sample was measured at 540 nm wavelength using the FLUOstar Omega microplate reader.

Example 14

The ability of anti-PD-L1 antibodies to modulate immune responsiveness was assessed using a mixed lymphocyte reaction (MLR). With this assay, the effects anti-PD-L1 antibodies on cell activation and the production of IL-2 were measured. The MLR was performed by culturing 10⁵ purified human CD4+ cells from one donor with 10⁴ monocyte derived dendritic cells prepared from another donor. To prepare the dendritic cells, purified monocytes were cultured with GM-CSF (1,000 U/ml) and IL-4 (500 U/ml) for seven days. Anti-PD-L1 or control antibodies were added to the allogeneic MLR cultures at 10 μg/ml unless stated otherwise. Parallel plates were set up to allow collection of supernatants at day 3 and at day 5 to measure IL-2 using a commercial ELISA kit (Biolegend). The antibodies used were the disclosed H6B1L, RSA1, RA3, RC5, SH1E2, SH1E4, SH1B11, and SH1C8 as compared to prior disclosed antibodies 10A5 (Bristol-Myers-Squibb/Medarex) and YW243.55S70 (Roche/Genentech) that were obtained via in-house production from prior-disclosed antibody sequences (U.S. Patent Application 2009/0055944 and U.S. Patent Application US 2010/0203056; the disclosure of which are incorporated by reference herein).

Production of IL-2 was enhanced by the addition of the anti-PD-L1 antibodies.

Example 3

Human primary sebocytes (Zenbio, RTP, NC) were plated at confluence on 96 well Scintiplates and allowed to adhere overnight. Cells were treated with the SCD1 inhibitor Compound A prepared in media containing the LXR agonist and insulin and cultured overnight. The DGAT inhibitor A922500 (2 μM) was included as a positive control. The following day ¹⁴C-acetate was added to each well and the plate was gently mixed. Cells were placed in the incubator at 37° C. for 4 hours total. After 2 hours of incubation the Cell Titer Blue (CTB) assay was started, 10 μl of CTB reagent was added to each well and incubated for the remaining 2 hours at 37° C. Following the 4 hour incubation, the RFU was determined using the SpectraMax Gemini EM under the following parameters: 560ex/590em with a 570 cutoff, top read. The medium was removed and cells were washed 3× with PBS. All of the PBS was removed from the wells and the plates were allowed to air dry. The plate was read in the MicroBeta TriLux counter and data was analyzed as CPM and normalized to CTB readout. Data is shown in FIG. 2.