Guava easycyte system

ROS production was measured by flow cytometry using dihydrorhodamine 123 (DHR123). Cells were preincubated for 20 minutes at 37 °C. The indicated agonists and DHR (2 µM) were then added for 15 minutes. To stop the reaction, cells were placed on ice, washed, and resuspended in Cell Wash (BD Bioscience) containing 0.5% PFA and 50 nM of the cell-impermeant far-red emitting nucleic acid stain Helix NP NIR (Biolegend) to gate for live cells. 10,000 live cells per condition were analysed on a Guava easyCyte™ System (Millipore, Burlington, MA, USA) for median fluorescence intensity. Agonist-mediated ROS production was expressed as percentage of the signal detected in untreated cells. The FPR2/ALX specific agonist WKYMVm was used as a positive control.

In flow cytometry, while all the procedures before the incubation step with the recombinant peptides were the same as those for CLFM, the culture medium-based recombinant peptides (5 μM) were incubated for 2, 4, 8, and 16 min. After incubation, the cells were washed carefully three times with PBS and harvested by intensive pipetting > 100 times. The harvested cells were centrifuged (200 RCF, 3 min) and washed with PBS. The procedure was repeated three times. Subsequently, the cells re-suspended in PBS were loaded into a flow cytometer (guava easyCyte System; Millipore, MA, USA) to acquire the Green-B fluorescence signals of EGFP of the recombinant peptides within the cells. For data analysis, the obtained data files were loaded on FlowJo software (Version 10; FlowJo LLC, OR, USA). Based on the forward scatter-versus-side scatter plots, the signals only for cells (> 4000) were collected, and the signal histograms and mean values were acquired using the software. To quantify the recombinant peptides adsorbed on or internalized in the cells, the mean values acquired at 2, 4, 8, and 16 min were subtracted from a mean value of the cells and fitted using the regression curves [ $\mathrm{\Delta }mean=a\left(1,-,b^t\right)$ ]. The increasing rate ( $k_{\mathrm{\Delta }\mathrm{m}}$ ) of the $\mathrm{\Delta }mean$ was determined at the initial point of the incubation as $-a\ln b$ .

Exponentially growing MDA-MB-231 and MCF 10A cells were seeded into 6-well plates at a density of 1.5 × 10⁵ cells/well in the presence of indicated concentrations of B02-iso or 0.1% DMSO (for control cells). After 24 h of incubation, cells were labeled with 10 µM Edu (Invitrogen; Waltham, CA, USA) for 1 h or left unlabeled in the control. Cells were harvested and centrifuged at 500× g, 4 °C for 5 min, and cell pellets were washed with PBS supplemented with 1% BSA, spun down, and fixed with 4% methanol-free formaldehyde (Pierce) in PBS for 15 min. Cells were washed with PBS containing 1% BSA, centrifuged 500× g for 5 min. Cell pellets were resuspended in PBS containing 0.1% Triton X-100 and incubated for 15 min for permeabilization. Cells were spun down, resuspended in freshly prepared Edu click labeling solution (1 mM CuSO₄, 1.25 µM Alexa Fluor 488 Azide (Invitrogen; Waltham, CA, USA), 50 mM ascorbic acid in PBS) and incubated for 30 min in the dark. Cells were washed twice with PBS containing 0.1% Triton X-100, resuspended in the same buffer, and analyzed for Edu incorporation by flow cytometry using the Guava easyCyte system (Millipore; Burlington, VT, USA) with the InCyte software (Millipore; Burlington, VT, USA).

The A172 cells were plated in 6-well plates in the standard incubation medium (4 × 10⁵ cells per well). 24 h after the plating, cells were exposed to ETM at concentration of 50 μM. To study the cell cycle, cells were collected by trypsinization, washed three times with PBS (pH 7.4), and then fixed and permeabilized by a dropwise addition of 70% aqueous ethanol precooled to −20 °C. The samples were kept for 4 h at +4 °C. Ethanol was removed from fixed samples by washing with PBS, then cells were resuspended in PI staining solution (0.01 mg·mL⁻¹ propidium iodide and 0.1 mg·mL⁻¹ RNase A in PBS) and incubated at room temperature for 30 min [43 (link)]. The samples were subjected to flow cytometry on a Guava easyCyte System (Millipore, Hayward, CA, USA) with Guava^® Cell Cycle Assay software (guavaSoft™ 3.1.1, Millipore, Hayward, CA, USA ). Fluorescence of propidium iodide was detected with a 488 nm excitation laser and a 695 nm emission filter.

Cells were harvested and fixed with 1% formaldehyde, permeabilized and blocked for 20 min (1X PBS, 0.1% tween 20, 5% FBS), incubated for 1 hr at room temperature with mouse 4G2 antibody and then 1 hr with goat anti-mouse IgG Alexa fluor 647 (Thermo Fisher Scientific) to detect ZIKV envelope protein (E protein). E protein fluorescence was measured with the Guava easyCyte system (Millipore) using the red laser (642 nm) for Figures 3 and 5 and Figure 3—figure supplements 1–3. Double staining was performed for Figure 6 to detect E protein and cellular FXR2 protein. E protein was detected using mouse 4G2 antibody and goat anti-mouse IgG Alexa fluor 488. FXR2 protein was stained using rabbit anti-FXR2 and goat anti-rabbit IgG Alexa fluor 647. Sample analysis, scatter plots of infected cells, mean fluorescence intensities and histograms were performed in FlowJo V10 software.

iPSC and iMSC surface antigens were analyzed by using flow cytometry. In total, 5 × 10⁵ cells were incubated with 1% bovine serum albumin (BSA) (Gibco) for 30 minutes to block non-specific antigens. The following conjugated monoclonal antibodies (BD Biosciences) were used at the concentration recommended by the manufacturer: CD29-PE, CD34-APC, CD44-FITC, CD45-FITC, CD73-PE, CD90-PE. CD105-FITC, CD133-PE, CD146-PE, and HLA-DR-PE. Non-specific fluorescence was determined by incubation of similar cell aliquots with isotype-matched mouse monoclonal antibodies (BD Biosciences). After two washes in 1% BSA, the cells were resuspended in 200 μL of 1% BSA and analyzed by using the guava easyCyte™ system (Millipore, Billerica, MA, USA).