Msd sector imager 2400 reader

Cytokine profiling was performed using Meso Scale Discovery (MSD) Multi-spot plates and an MSD Sector Imager 2400 reader (Meso Scale Discovery). Using the MSD 10-Plex Mouse Proinflammatory Panel I, a total of 10 cytokines were measured simultaneously (IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, KC/GRO, and TNF-α) according to manufacturer’s instructions. Briefly, tumor and mammary gland lysates were diluted 1:10 in supplied assay diluent. Sample lysate, 50 μl, was added in duplicate to the appropriate wells in a 96 well plate. The array was incubated for 3 h at room temperature, while shaking. The array was then washed with phosphate-buffered saline plus 0.05% Tween 20, and 25 μL of detection antibody reagent was added and incubated for 2 h while shaking. The array was washed, detection buffer added and results read using an MSD Sector Imager 2400 incorporating a charge-coupled device. Sample cytokine concentrations were determined with Discovery Workbench software (Meso Scale Discovery).

VEGF expression signals from archival FFPE tissues were measured as previously reported [14 (link)]. Briefly, five microliters of protein extract from FFPE tissue specimen, at predetermined protein concentrations, were added to Meso Scale Discovery (MSD, Gaithersburg, MD) Multi-Spot™ plates (MA2400 96 HB Plate). The plate was allowed to dry at room temperature for 90 min, and the plates were subsequently further incubated for 30 min at 37°C. The antigen-coated plates were preincubated with 5% BSA in PBST for 60 min at RT before primary antibody reactions. Anti-VEGF (Neomarkers) and anti-GAPDH (Calbiochem) were diluted 1:200 and 1:1000 with 5% BSA in PBST, and then incubated overnight at 4°C. After washing with PBST, the plates were incubated for 1 h with goat anti-mouse SULFO-TAG™ antibodies at a dilution of 1:1000 (0.5 μg/ml) with 5% nonfat milk in PBST. The plates were then aspirated and washed three times with PBST. Finally, MSD-T read buffer was added to the plates and they were read on the MSD Sector Imager 2400 reader (Meso Scale Discovery). BSA coated wells were included on each plate as a control for non-specific binding effects. The values from non-specific wells were subtracted from all standards samples to calculate actual value. Two independent experiments were performed with triplicates.

Total proteins were extracted from three 10 μm frozen tissue sections using T-PER buffer (Pierce Biotechnology) with proteinase inhibitor cocktail (1 tablet/25 ml, Roche). Two μg protein extract from frozen tissue specimen was added to Meso Scale Discovery (MSD, Gaithersburg, MD) Multi-Spot™ plates (MA2400 96 HB Plate), the plate was allowed to dry at room temperature for 90 min, and the plates were subsequently further incubated for 30 min at 37°C. The antigen-coated plates were preincubated with 3% nonfat milk in PBST for 60 min at RT before primary antibody reactions. Anti-CSPG4 (TP41.2) and anti-Actin (Abnova, mouse, clone 3G4-F9) were diluted 1:1000 and 1:5000 respectively, with 3% BSA in PBST, and then incubated overnight at 4°C. After washing with PBST, the plates were incubated for 1 h with goat anti-mouse SULFO-TAG™ antibodies at a dilution of 1:2000 (0.5 μg/ml) with 5% nonfat milk in PBST. The plates were then aspirated and washed three times with PBST. Finally, MSD-T read buffer was added to the plates and they were read on the MSD Sector Imager 2400 reader (Meso Scale Discovery). BSA coated wells were included on each plate as a control for non-specific binding effects. Signal was normalized to actin expression and is expressed as relatively fold over background (water).