Cytoflex benchtop flow cytometer

The experiment was performed as described in Fig 3A. Cells were grown in SC-Complete media overnight at 30°C in the shaking incubator and diluted in the morning to OD600 = 0.3. 35 mM HU was then added to the culture for 4 h, then washed out and cells were allowed to progress through the cell cycle for 6 h. Samples were taken at 0, 2, 4, and 6 h after release, fixed with 70% cold ethanol and incubated overnight at 4°C. Cells were then pelleted and washed with 1 ml sodium citrate buffer, pH 7.4. Afterwards, 1 ml sodium citrate buffer and 25 μl of the 10 mg/ml RNase A were added to the pellets and cells were incubated overnight at 37°C. Cells were pelleted, washed with sodium citrate buffer then treated with 10 mg/ml proteinase K for 2 h at 37°C. Pellets were then resuspended in 500 μl sodium citrate buffer and 6 μl of 1 mg/ml propidium iodide. FACS analysis was performed using The Beckman Coulter Life Sciences CytoFLEX benchtop flow cytometer. Files were processed using the FlowJo software and % of cells in 1C, 2C, and 4C were determined using the same software.

Standard plate colony counting method was used to determine the number of bacteria in the Lb. casei Zhang culture (found to be 10⁹ colony-forming units [CFU]/mL), which was then serially diluted to 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, and 10⁹ CFU/mL, respectively. Then, 3 mL of each bacterial dilution was washed and resuspended in the same volume of phosphate-buffered saline. Then, each tube was split into three 1 mL aliquots. The first aliquot was processed for FCM using a CytoFLEX benchtop flow cytometer (Beckman Coulter, Inc., Indianapolis, IN, USA); cells were incubated at 37 °C with CFDA (10 μL of 5 mmol/L) for 15 min followed by incubation with PI (10 μL of 1.5 mmol/L) for another 15 min in the dark. The second aliquot was used as an FCM negative control (no addition of fluorescent dyes). The third aliquot was used for plate colony counting. The plate colony counting and FCM counting methods were compared with respect to the detection range and bacterial density.

Purified exosomes were incubated with 10 μg/ml NS17‐FITC (produced in house), anti‐CD81‐APC (Almog Diagnostic, Shoham, Israel) or were left unstained for 60 min on ice protected from light. Mix of stained beads in different sizes (100, 200, and 500 nm) were used as an internal control to verify the size of the nanoparticles. Analysis was performed using CytoFlex Benchtop Flow Cytometer (Beckman Coulter Life Sciences).

Cells were harvested, fixed in pre-chilled 70% ethanol at 4°C overnight, washed twice with PBS, and incubated in PI/RNase staining buffer (BD 550825) at room temperature for 15 minutes. Cell numbers in different cell cycle stages were counted by CytoFLEX benchtop flow cytometer (Beckman Coulter), and the data were analyzed by ModFit LT 4.1 software.

HepG2 cells were plated in a 12-well plate at 4 × 10⁵ cells/well for 12 h. After incubation for 24 h with verapamil dissolved in PBS, Vera@pullulan, and Vera@CLCMP patches at the indicated concentrations, the adherent and floating cells were collected, washed with cold PBS, and stained using the Dead Cell Apoptosis kit with annexin V-FITC and propidium iodide (Thermo Fisher Scientific, Waltham, MA, USA). Data was analyzed using a CytoFLEX benchtop flow cytometer (Beckman Coulter, Fullerton, CA, USA).

Lysed cell suspension was centrifuged at 500xg for 5 minutes, washed twice with phosphate-buffered saline (PBS) and finally, obtained lysed cells were suspended in PBS. Whole blood was assessed for the distribution status of platelets, neutrophils, monocytes and T-T-lymphocytes using 13-color flow cytometer CytoFLEX system (Beckman Coulter Life Sciences CytoFLEX benchtop flow cytometer). Obtained cell suspension was incubated for 30 min in the dark at room temperature with anti-human monoclonal antibodies for identification of cellular subsets: CD-42b-PE (Beckman Coulter Life Sciences, IM1417U) for platelets, CD14-PC7 (Beckman Coulter Life Sciences, A22331) for monocytes, CD66b-APC-Alexa Fluor 750 for neutrophils (Beckman Coulter Life Sciences, B08756), and CD3-ECD (Beckman Coulter Life Sciences, IM2705U) for T-T-lymphocytes. Following incubation, cells were washed with PBS to be suspended in 300 μL and analyzed by flow cytometry for gating platelet-specific CD42b-PE positive population, neutrophil-specific CD66b-APC-Alexa Fluor 750 positive population, monocytes-specific CD14-PE positive population, and T-T-lymphocytes specific CD3-ECD positive population. Data was acquired for 20,000 events and analyzed using CytExpert software to assess the percentage and mean fluorescence intensities (MFIs) of cellular subsets.