Lsrfortessa sorp cell analyser

Activity of the lsr promoter was assayed using a plasmid-based egfp reporter that contains the 217 nucleotide region upstream of the lsrA gene. Samples for flow cytometry were prepared as described above, diluted 1:20 in tethering buffer (10 mM KH₂PO₄, 100 μM EDTA, 1 μM L-methionine and 10 mM lactic acid, pH=7.0) and fluorescence was measured with BD LSRFortessa SORP cell analyser (BD Biosciences, Germany). Before the measurements, cell aggregates were dispersed by vigorous mixing.
The same reporter transformed in ΔluxS strain was used as a biosensor to quantify levels of AI-2 in supernatants. Cell-free supernatants were prepared by filtration of liquid cultures through 0.2 μm filter, and 20 μl aliquots of the reporter strain (OD₆₀₀=0.5) were added to each sample followed by 40 min incubation at 37 °C. The reporter was calibrated using defined concentrations of synthetic DPD/AI-2.

Treatments were added for 48 or 72 h to 50,000 cells per well in 96-well plates. Following treatment, the wells were washed once with 100 μL of cold DBPS, centrifuged at 500g at 4 °C for 1 min and resuspended in 50 μL of DPBS containing 1 μL/mL of LIVE/DEAD^® Fixable Near-IR Dead cell stain for 633/635 nm to stain dead cells following manufacturer’s instructions (Thermo Fisher Scientific Inc. (NSYE: TMO)), and fixed in 100 μL of 0.5% PFA. High throughput flow cytometry was performed directly from the 96-well plates using a BD LSRFortessa™ SORP cell analyser using the BD™ High Throughput Sampler Option (HTS)-LSRFortessa microplate adaptor and acquisition was performed using the following detection settings: Near-IR from the Red laser 780/60-A [642 nm], mCherry from the Yellow-Green laser 610/20-A [561 nm] and GFP from the Blue laser 530/30-A [488 nm]. Reactivation from latency was measured only in live single-cells by negative gating of dead cells, followed by gating on mCherry⁺ (transduced cell lines only), and then GFP⁺ or GFP⁻. Reactivation from HIV-1 latency was quantitated as the percentage of GFP positive cells and as the mean fluorescent intensity (MFI) of the GFP signal.