Lsr 2 fortessa cytometer

Memory B cells were stained with 5 μM Cell Trace Violet (ThermoFisher Scientific, Paisley, UK) as directed in the datasheet, and incubated overnight at 37°C 5% CO
₂. The cells were then washed and cultured using the optimal DoE conditions. For flow cytometry experiments cells were then stained with Aqua Live/Dead cell viability dye (BD Biosciences, Oxford, UK) as per manufacturer’s instruction. Cells were then stained with the phenotyping panel shown in
Table 2. The cells were analysed using an LSR Fortessa II cytometer (BD Biosciences) at baseline, day 5 and day 10 of culture. The gating strategy used can be seen in
Supplementary Figure 1. Purity of CD27
⁺ memory B cells following isolation by magnetic selection was also determined using this panel. For FACS, memory B cells were sorted based upon IgD and IgM expression into 4 sub-populations (IgD
⁺ IgM
^-, IgD
⁺ IgM
⁺, IgD
^- IgM
⁺, IgD
^- IgM
^-) as shown in
Supplementary Figure 1. Cell sorting was carried out using a BD FACSAria III.

CCF-4 assay was conducted as previously described [28 (link)], with minor modifications. Briefly, 1 x 10⁶ THP-1 cells were seeded on 12-well tissue culture plates and treated with 33 nM PMA for three days. Cells were then washed with media and then infected with single-cell suspensions of tdTomato-expressing wildtype, ΔESX-1, or ΔmmpL7 M. marinum at a MOI of 1 for 4 h at 33°C in EM medium. 24 hours post infection cells were stained with Fixable Viability Dye eFluor660 (eBioscience). Cells were then harvested and stained for 1h at room temperature with 8 μM CCF4-AM (Invitrogen) in EM medium supplemented with 2.5 μM probenecid. Finally, cells were fixed overnight at 4°C in 4% (wt/vol) paraformaldehyde. Cells were analyzed in an LSRFortessa II cytometer, using FACSDiva software (BD Biosciences). At least 40,000 events per sample were collected. Data were analyzed using FlowJo (Treestar). Permeabilization percentage among infected monocytes was calculated using a region defined by an increased 450 nm signal (indicating CCF4 dye cleavage and, therefore phagosomal permeabilization) relative to uninfected cells in the same sample. Events in this region were designated “permeabilized”. Percent permeabilization was then calculated as a ratio of events in the permeabilized field over total live and infected cells.

Positive results in the ELISpot assay were validated by ICS for IFN‐γ as described previously.³⁵ The pre‐cultured cells were restimulated with the peptides showing a positive result at a concentration of 10 μg mL⁻¹ for 16 h at 37°C and 5% CO₂. After one hour, Brefeldin A (Sigma‐Aldrich, St. Louis, MO, USA) in a final concentration of 5 μg mL⁻¹ was added to inhibit cytokine secretion.
The cells were stained with Zombie NIR fixable viability dye (BioLegend, San Diego, CA, USA) and the following fluorochrome‐conjugated monoclonal antibodies on the cell surface: anti‐CD3 (clone UCHT1, Alexa Fluor 700), anti‐CD4 (clone SK3, BV510), anti‐CD8 (clone RPA‐T8, PerCP‐Cy5.5), anti‐CD14 (clone 63D3, APC‐Cy7) and anti‐CD19 (clone HIB19, APC‐Cy7). After fixation and permeabilisation using the FoxP3 transcription factor staining buffer set (eBioscience, Thermo Fisher Scientific), the cells were stained for intracellular IFN‐γ using a monoclonal anti‐IFN‐γ antibody (clone 4S.B3, PE‐Dazzle594). All antibodies were purchased from BioLegend. The cells were acquired on a LSRFortessa II cytometer (BD) using FACSDiva version 8 for Windows (BD). The full gating strategy is reproduced in Supplementary figure 14.

The cytotoxicity of CBD and CBG was evaluated by flow cytometry with propidium iodide according to the protocol of Riccardi and Nicoletti (2016) [30 (link)] with minimal modifications. For all tests, CBD and CBG were used with a purity of 99.9%. CBD and CBG were reconstituted in dimethyl sulfoxide (DMSO, Thermo Scientific™) at a concentration of 0.01% and evaluated between 0.5 µM and 20 µM based on previous reports in the literature [31 (link)]. CBD and CBG compounds were tested at concentrations of 0.5, 1, 3, 10, and 20 µM at 3, 6, and 12 h. The positive control was 100 µM hydrogen peroxide, and the negative control was DMSO (0.01%).
A suspension of 1 × 10⁶ cells (PLF) in 1 mL PBS in 12 × 75 mm tubes (Biolife, Bothell, WA, USA) was centrifuged at 200× g for 5 min at room temperature, the PBS was removed, and the cell pellet was resuspended in 1 mL of fluorochrome solution (Becton Dickinson, Franklin Lakes, NJ, USA). Tubes were placed in the dark at 4 °C before flow cytometry for at least 1 h and no more than 24 h. The LSR Fortessa II cytometer (Becton Dickinson) with a 488 nm laser line was used for excitation. Red fluorescence (>600 nm) and dispersion were measured, collecting at least 20,000 events. The FlowJo^® 8.7 software (Tree Star, Inc., Ashland, OR, USA) was used to carry out the analysis.

Cytokine quantification was performed using the LEGENDplex kit (BioLegend, San Diego, CA, USA) Ref 740930. CBD and CBG at 0,5, 1, 3, 10, 20 uM concentrations were added to PLF cultures at 3, 6, and 12 h and the production of IL-2, CXCL10, IL-1β, TNF-α, and CCL2, IL-17 A, CXCL8, IL12p70, IL-6, IFN-γ, IL-10, TGF-β, and IL-4 were measured. The negative control was cell culture supernatant of periodontal ligament fibroblasts in base medium (DMEM) with DMSO at a concentration of 0.01%. The LSR Fortessa II cytometer (Becton Dickinson) was used, and the results were generated by the LENGENDplex Data Analysis Software v8.0 (BioLegend, USA). The protocol used was the one provided by the manufacturer. The cytometer reading was simultaneous for all conditions [32 (link)].