S3e cell sorter

CD138⁻ cells were isolated by two methods using the S3e™ Bio-Rad Cell Sorter and magnetic beads. MM.1R, and RPMI 8226 cells were washed and resuspended in cold PBS with 3% FBS. Then cells were stained with cell viability dye VivaFix498/521 and mouse anti-human CD138 Alexa Fluor 647 and incubated in the dark on ice for 25 min. After washing twice, cells were sorted using S3e™ Bio-Rad following the manufacturer’s protocol (Itoua Maïga et al., 2014 (link)). Alternatively, CD138 magnetic beads were used to isolate CD138⁺cells. Human MM cells MM.1R and RPMI 8226 cells were fractioned by a MACS separator, using CD138 magnetic beads. After washing cells from the media, cells were resuspended in 0.5% MACS buffer and incubated with CD138 microbeads at 4°C for 15 min. After washing, CD138⁻ cells were isolated using an MS column according to the manufacturer’s protocol (Pandey et al., 2017 (link)). Similarly, CD19⁺CD138⁻, and CD19⁺CD27⁺, CD38⁻ cells were isolated using S3e™ Bio-Rad Cell Sorter following the manufacturer’s protocol. Briefly, after suctioning out media, cells were washed with PBS that contains 3% FBS. Cells were incubated with respective antibodies in the dark on ice for 25 min and then washed twice with PBS and sorted using S3e™ Bio-Rad.

Expressed yeast
cells were analyzed using a BD Accuri C6 Plus Flow Cytometer (BD Life
Sciences, USA) S3e Cell Sorter device (BioRad, USA). The gating strategy
is shown in Figure S9. Green fluorescence
channel (FL1-A) was used to record eUnaG2 or FITC signals representing
expression positive cells, and a far-red fluorescent channel (FL4-A)
recorded CF640R stained proteins binding signals. No compensation
was applied. Negative cells, EBY100 cells without plasmid or nonlabeled
cells, were used to determine the negative population and set quadrant
gating. Quadrants were used to divide the gated cell population into
four plots showing negative (LL), nonspecific (UL), expression (LR),
and binding (UR) populations.
FACS experiments were performed
using a S3e Cell Sorter (BioRad, USA). Cells with surface-expressed
proteins, detected via eUnaG2, DnbALFA cocultivation
labeling, or c-myc antibody labeling, were incubated for 1 h at 4
°C with bait protein and mixed using a lab rotator (5 rpm). Before
the sorting, samples were collected by centrifugation (3000 g, 3 min),
1 to 3 times washed with ice-cold PBSB buffer (1 mL), and passed through
a cell strainer (40 μM, SPL Life Sciences, Korea).

V. cholerae WT and the indicated var mutant strains were grown for 5 h in the presence of 1 mM As^V, and then aliquots (1 mL) from each culture were pelleted, washed once, and resuspended in PBS (100 μL) to measure membrane potential and ROS.
To measure membrane potential in V. cholerae cells, we used the BacLight bacterial membrane potential kit (Invitrogen). V. cholerae samples resuspended in 100 μL PBS were incubated for 15 min at 20°C in the dark with the dye 30 μM 3,3′-diethyloxacarbocyanine iodide (DiOC₂). Next, cells were washed twice with PBS and resuspended in 2 mL of PBS, and then the fluorescence emitted from treated cells was measured with a Bio-Rad S3e cell sorter at an excitation wavelength of 488 nm and an emission wavelength of 525/30 nm (for green) or 655 nm (for red).
ROS measurements were performed using the CellROX green reagent for oxidative stress detection (Thermo Fisher). Washed V. cholerae cells were supplemented with CellROX reagent at a final concentration of 5 μM and were incubated for 30 min at 37°C. Next, cells were washed twice with PBS and resuspended in 2 mL of PBS, and then the fluorescence emitted was measured with a Bio-Rad S3e cell sorter at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.