Propidium iodide solution

Cells were plated in 6-cm dishes and grown overnight. Twenty-four hours after growth medium was changed to the leucine-competent or -deficient, the cells were harvested, washed with PBS and fixed with 70% ethanol overnight at −20 °C. After rinsed with PBS containing 3% bovine serum albumin, the cells were resuspended in PBS with 50 μg/ml propidium-iodide solution (Sigma-Aldrich) and 10 μg/ml RNase A (Sigma-Aldrich) for 30 minutes on ice. The stained cells were counted by a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA).

NET cells were seeded and grown in 6-well plates overnight and then treated with various doses of trametinib (TMT212), SCH772984, or trametinib (TMT212) plus ribociclib (LEE011) or SCH772984 plus ribociclib (LEE011) for 72 h. Next, the cells were harvested using 0.05% trypsin, centrifuged and washed in phosphate-buffered saline (PBS), then resuspended in the propidium iodide solution (Sigma-Aldrich, Taufkirchen, Germany) for flow cytometric analysis and quantification of the cell cycle distribution with BD Accuri C6 Analysis software (Biosciences, Heidelberg, Germany).

Flow cytometry was used to quantitate the production of T1P by the EPEC strains. These strains were grown overnight in LB media at 37°C or 26°C and the optical density adjusted to an OD₆₀₀ of 1.1. Forty-five μl aliquots were incubated for 1 h on ice with 25 μl of anti-T1P antibodies at a dilution of 1:500. After three gentle washes with PBS, the bacteria were resuspended in 25 μl of a 1:500 dilution of goat anti-rabbit IgG (H + L) Alexa Fluor conjugate (Invitrogen, Carlsbad, CA). After 1 h incubation at 4°C, the bacteria were gently washed three times with PBS and resuspended in 800-μl final volume of PBS. For the analysis, the bacteria were labeled with 3 μl of a propidium iodide solution (Sigma, St. Louis, MO). Propidium iodide (red) was visualized through a 42 nm band pass centered at 585. These experiments were repeated in triplicate. The FITC (green) fluorescence emission was collected through a 30 nm band pass filter centered at 530 in which 50,000 events were measured. The samples were analyzed at the ARL Biotechnology/ACCC Cytometry Core Facility at the University of Arizona, by using a FACScan (Becton Dickinson, Franklin Lakes, NJ).

SyS cells were dissociated with trypsin and fixated in 70 % ethanol after treatment with different concentrations of palbociclib at different time points. Propidium iodide solution (Sigma-Aldrich, 50 μg/ml in PBS) was added to the fixated cells to achieve DNA staining, and cells were treated with RNAse A (Qiagen, 100 μg/ml). All samples were measured using flow cytometry (CyAn ADP Analyzer, Beckman Coulter) and analyzed by FlowJo software (version 10). Hereby viable single cells were selected by gating.

RAW264.7 cells were seeded at 0.2 x 10⁶ cells/well in 6-well plates, cultured in DMEM with 10% FBS at 37^°C for 12 h. After 12 h, cells were infected with Mtb strains at MOI 5 and incubated for 12, 24, 36 and 48 h. Prior to harvesting, the cells were pulse labelled with bromodeoxyuridine (BrdU) for 45 min. Uninfected cells and unlabelled BrdU cells were used as negative controls. The harvested cells were stained with the live/dead stain (Ex ~633 nm/ Em ~780 nm, Invitrogen), washed and fixed in ice cold 70% ethanol for 12 h. After washing with PBS, the cells were treated with 2N HCl for 20 min to expose BrdU labeled DNA and the acidic pH was neutralized with 0.1 M sodium borate buffer, pH 9.0. The cells were then permeabilized with 1X permeabilization buffer (BD Biosciences) containing 0.3% Triton X-100 to ensure nuclear membrane permeabilization and washed once with PBS. Cells were labelled with APC-conjugated anti-BrdU antibody (Biolegend) for 1 h on ice. After washing by centrifugation, the cells were resuspended in 0.2 ml of propidium iodide solution (Sigma), and allowed to incubate for 30 min on ice. Cell cycle analysis was performed by data acquisition with Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo Vx.0.7. Two independent experiments were performed in triplicate.

To measure surface expression of the VHH, 100 μL of cell suspension (∼5 × 10⁷ cells) was incubated with 5 µL of 50 μg/mL anti-Myc-Tag (9B11) Mouse mAb (Alexa Fluor 488 conjugate) (Cell Signaling Technology, UK) at 4 °C in the dark for 30 min. The cells were washed twice and resuspended in 50 μL PBS. To test for cell viability, 1 µL of 1 mg/mL propidium iodide solution (Sigma-Aldrich, USA) was added. Cells were diluted 200-fold in PBS and analyzed using an Attune NxT flow cytometer (Thermo Fisher Scientific, USA) with collection of at least 100,000 events. Uninduced cells were used as a negative control and for gating. Data analysis was carried out using the FlowJo vX.0.7 software (FlowJo, LLC, USA).
For quantification of VHH expression, the same protocol was used, but the amount of the labelling antibody was varied (0–10 pmol) and the correlation curve between geometric means and the antibody amounts was fitted using a non-linear least squares regression model in Prism 7 software (GraphPad, USA) to estimate the concentration at which binding of the anti-Myc-Tag antibody was saturated.