Glycolytic burst assay

Seahorse XF-96 cell culture plates were seeded with 2 x 10⁵ cells per well concentrated in the center of the plate with two rows of 200 μL ccH2O layering the outside of the plate (4 rows down and 8 columns across, 32 wells total in the middle of the plate) to act as a micro humidifier for evaporation considerations. These considerations are warranted for the Seahorse XFe96 machine and not the Seahorse XF-Pro. Cells were plated directly after harvest on Day 6 of differentiation. At indicated time points, DCs were analyzed in XF running buffer (unbuffered RPMI 1640, 5 mM glucose, 10% FBS 1 +/- 500 μM SEITU) per the manufacturer’s instructions to obtain real-time measurements of OCR and ECAR. Where indicated, ECAR and/or OCR were analyzed in response to 100 ng/mL LPS. All drug dilutions were done in plain XF unbuffered RPMI. The program on Wave went as follows: 6 baseline read cycles, injection 1 (LPS), 20 read cycles, measurement period 2, 92 read cycles. Each read cycle was 3 minutes of mixing and 3 minutes of measuring for a total time of 12 hours. We note here that around 11 hours after the run has started, readings become erratic due to the chamber being non-humidified, therefore, we chose to only show the first 10 hours of the 12-hour period.