Biotek cytation 1 cell imaging multi mode reader

Mitochondrial oxygen consumption rate (OCR) was measured using a Mito Stress Test (31 (link)) and Seahorse XFe96 technology (Agilent Technologies, Santa Clara, CA). Naïve and IL-12-differentiated CD4⁺ T cells were seeded on Cell-Tak (Corning, Corning, NY)-coated 96-well Seahorse plates in triplicate at a density of 3 x 10⁵ cells/well within 200 ul, at a concentration of 1.5 million cells/ml, and maintained under standard cell culture conditions (5% CO₂, 37⁰C). Cells were incubated in XF Assay Medium (pH 7.4) with sodium pyruvate (1 mM), L-glutamine (2 mM), and glucose (10 mM) at 37°C without CO₂ for 1 hr before measuring T cell OCR with an XFe96 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA) according to manufacturer’s instructions. Respiratory parameters were assessed by the sequential addition of oligomycin (1 μM), carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP; 2 μM), and rotenone/antimycin A (0.5 μM). OCR measurements were normalized to cell count obtained by staining nuclei with Hoescht dye (2 μM; ThermoFisher Scientific) and visualizing on a BioTek Cytation 1 cell imaging multi-mode reader (BioTek, Winooski, VT).

Biotek Cytation 1 Cell Imaging Multi-Mode Reader (BioTek Instruments Inc., Winooski, VT, USA) and Gen5 Microplate Reader and Imager Software (catalog no. GEN5, BioTek) were used to acquire data. The filter cubes used to image the cells were DAPI (Cat #1225100, BioTek), GFP (Cat #1225101, BioTek), and RFP (Cat #1225103, BioTek). The Gen5 Spot Counting Module (GEN5SPOT, BioTek) was used to quantify foci in treated nuclei. For each of the 4 biological replicates to be analyzed, 1500 nuclei were randomly selected. The mean number of spots in the no treatment control wells was normalized to 1; all other treatments were normalized to this value.

For the living cell experiment, we assayed the yield by sorting one cell into each well of 1536‐well plates. Jurkat cells were stained by CFSE and Hoechst 33342. A mixture of stained cells and unstained cells with a volume ratio of 1 : 1 was diluted in PBS buffer (cell concentration: 10⁵–10⁶ cells·mL⁻¹). White 1536‐well plates (Corning, Corning, NY, USA) were prepared by adding 5 μL of PBS to each well. After sorting, the plate was removed and centrifuged to keep the cell at the bottom of the well. Each well of a 1536‐well plate was scanned with a BioTek Cytation 1 Cell Imaging Multi‐Mode Reader (BioTek) individually.
The purity assay was performed as follows. CFSE‐stained Jurkat cells were mixed at 1 : 1 with unstained cells to obtain a cell suspension (1 × 10⁷ cell·mL⁻¹). Changing the drop delay value, 30 000 negative and positive cells were collected using each different drop delay value. Then, the cell purity was detected by flow cytometry.