V. parahaemolyticus was inoculated in 96-well polystyrene plates (SPL life science, South Korea) without shaking at 30°C for 24 h with or without the indoles. The biofilms were stained with 100 μL of pre-warmed PBS containing CFSE stain (carboxyfluorescein diacetate succinimidyl ester) (Invitrogen, Molecular Probes, Inc., Eugene, OR, United States) for 20 min at 37°C (final concentration, 5 μM) and washed three times with PBS. The static biofilm plates were visualized by excitation using Ar 488 nm (emission wavelengths 200–550 nm). The cells were visualized by confocal laser microscopy (Nikon Eclipse Ti, Tokyo, Japan) using a 20× objective. DMSO was used as the control. Color confocal images were visualized using NIS-Elements C version 3.2 (Nikon eclipse). For each experiment, at least 10 random positions in three independent cultures were chosen for microscopic analysis (Sethupathy et al., 2020 (link)).
Cfse stain
Manufactured by Thermo Fisher Scientific
Sourced in United States
CFSE stain is a fluorescent dye used for cell proliferation analysis. It binds to cellular proteins, allowing the tracking of cell division through successive generations.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using cfse stain
1
Biofilm Formation Dynamics of V. parahaemolyticus
2
MDSC Isolation and T-cell Proliferation
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MDSC sorting and T-cell proliferation were described previously [43 (link),44 (link)]. An MDSC isolation kit (Miltenyi Biotech, Germany) was used according to the manufacturer's instructions to obtain splenic PMN-MDSCs and M-MDSCs. Normal splenocytes were incubated with carboxyfluorescein succinimidyl ester (CFSE) stain (Invitrogen, USA), and activated with anti-CD3 (5 μg/mL, eBioscience, CA) and anti-CD28 (5 μg/mL, eBioscience, CA) antibodies in Roswell Park Memorial Institute Medium (RPMI) 1640. Then PMN-MDSCs/M-MDSCs were cocultured with splenocytes (MDSCs: splenocytes = 1:2, 1:4 and 1:8) in 96-well flat-bottom plates. CFSE is divided equally among daughter cells with each division; therefore, T-cell proliferation could be assessed by flow cytometry 72 h later. All experiments were run in triplicate.
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