Tnfα elisa

TNFα protein concentrations were measured using a TNFα ELISA (Thermo Fisher scientific, Pittsburgh, PA). Into all wells, 100 μg protein was loaded, with analyses based on a standard curve. A human IL-1β ELISA (R and D system, Minneapolis, MN) was used to measure levels of the pro-inflammatory cytokine, IL-1β, in cell lysates. Equal protein was loaded (100 μg) into all wells to allow for comparisons based on OD. Manufacturer’s instructions were followed for all ELISA, except extended incubation of samples and antibody reagents overnight at 4 °C.

RAW264.7 cells (20,000/well) were seeded on a 96 well plate and allowed to attach overnight. The following day the cells were treated with PBS or 0.4 μg GO-EV for 3 h. The media was collected and centrifuged at 1,500 rpm for 10 min. The supernatant was collected and utilized to perform a mouse 31-plex cytokine and chemokine panel (Eve technologies, Alberta, Canada). For TNF-α assays, cells were treated with PBS, 0.4 μg GO, sGO, GO-EV, sGO-EV or az-EV for 24 h. Samples were diluted in PBS. TNF-α assays were performed by a high sensitivity TNF-α ELISA (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s protocol, using a FLUOstar Omega plate reader (BMG Labtech, Germany) to measure absorbance. A four-parameter fit standard curve was generated using RStudio. For cytokine assays, four technical replicates were included for each treatment condition.

A cleaved caspase 3 ELISA (Cell Signaling, Danvers, MA) was used to measure levels of the active apoptotic marker in rMC-1 cells, while phosphorylation of IRS-1 on serine 307 was measured with ELISA (Cell Signaling, Danvers, MA) with equal protein loaded (50 μg) for both ELISAs to allow for analyses using optical density measurements. By loading equal protein into the cleaved caspase 3 and IRS-1^Ser307 ELISA, we eliminated bias for transfection efficiency or cell numbers. TNF-α protein concentrations in the cell lysates were measured using a TNF-α ELISA (ThermoFisher, Pittsburgh, PA).

TNFα protein concentrations were measured in the cell lysate after salicylate treatment using a TNFα ELISA (ThermoFisher, Pittsburgh, PA) according to the manufacturer’s instructions. Equal protein levels were added to the ELISA to ensure cell numbers did not affect concentrations. A cleaved caspase 3 ELISA (Cell Sigaling, Danvers, MA) was used to measure levels of active apoptotic marker in retinal cells. Equal amount of protein was loaded for cleaved caspase 3 ELISA to allow for analysis using the optical density measurements.

A cleaved caspase 3 ELISA (Cell Signaling, Danvers, MA) was used to measure levels of the active apoptotic marker in whole retinal lysates. TNFα protein concentrations were measured using a TNFα ELISA (ThermoFisher, Pittsburgh, PA). For cleaved caspase 3 ELISA analyses, equal protein was loaded (60 μg) into all wells to allow for comparisons based on optical density (O.D.) For the TNFα ELISA, 100 μg protein was loaded into all wells, with analyses based on a standard curve. For phospho-IRS1 ELISA (Cell Signaling, Danvers, MA) analyses, 100 μg protein was loaded into all wells to allow for comparisons based on O.D.

TNFα protein concentrations were measured using a TNFα ELISA (ThermoFisher, Pittsburgh, PA). 100 μg protein was loaded into all wells, with analyses based on a standard curve. The manufacturer's instructions were followed.