Bacteria were grown to stationary phase at 37 °C in LB medium adjusted to pH 5.0. A 1-ml aliquot of culture was then centrifuged for 5 min at 2000 × g, and the resulting pellet resuspended in 1 ml of LB medium adjusted to either pH 2.5 or pH 5.027 (link)28 (link). The suspension was then incubated for 30 min at 37 °C with shaking at 220 rpm. Bacteria were collected by centrifugation as described above, and washed three times with PBS. Cells were then stained for 15 min using a BD cell viability kit, and measured using a BD FACS flow cytometer. The data were analyzed using CellQuest software.
Cell viability kit
Manufactured by BD
Sourced in United States, Italy
The BD Cell Viability Kit is a laboratory equipment product designed to measure the viability of cells. It provides the core function of assessing the number of live and dead cells within a sample.
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Lab products found in correlation
Liquid counting beads, by BD (4 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
Lb agar plate, by Merck Group (2 mentions)
Gaspak ez anaerobe pouch system, by BD (2 mentions)
Liquid chopped meat broth, by Hardy Diagnostics (2 mentions)
Accuri c6 flow cytometer, by BD (2 mentions)
Fc block, by BD (2 mentions)
Facscan flow cytometer, by BD (2 mentions)
Facsflow, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Facs flow cytometer, by BD (1 mentions)
Butyric acid, by Merck Group (1 mentions)
Ruminococcus bromii, by American Type Culture Collection (1 mentions)
Celltiter glo, by Promega (1 mentions)
Sp6800 spectral analyzer, by Sony (1 mentions)
Annexin 5 apoptosis detection kit apc, by Thermo Fisher Scientific (1 mentions)
Encapsulator b 390, by Büchi (1 mentions)
Mc 813 70, by BioLegend (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
Anaerocult a, by Merck Group (1 mentions)
36 protocols using cell viability kit
1
Bacterial Viability Assay at Low pH
2
Cultivation of Bacterial Strains
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Salmonella enterica subsp enterica serovar Typhimurium (ATCC 35986) was grown under aerobic conditions at 37°C for 1–2 d on LB agar plates (Sigma-Aldrich). Growth of anaerobic bacteria was performed using a BD GasPak EZ Anaerobe Pouch System (BD Diagnostics). Prevotella stercorea (DSM No. 18206) was grown for 5–7 d under anaerobic conditions in liquid-chopped meat broth (Hardy Diagnostics) supplemented with 1% Trace Minerals (ATCC), 1% Vitamin Supplements (ATCC), 0.05% Tween 80, 29.7mM acetic acid, 8.1 mM propionic acid and 4.4 mM butyric acid (Sigma-Aldrich). Ruminococcus bromii (ATCC# 27255) was grown under anaerobic conditions at 37°C for 1–2 d in liquid-chopped meat broth (Hardy Diagnostics). The long-term stock of Bifidobacterium longum subsp infantis (ATCC 15697) was grown under anaerobic conditions at 37°C for 2–3 d in liquid-chopped meat broth (Hardy Diagnostics) and the working stock was grown on Brucella plates (Teknova) under anaerobic conditions at 37°C for 2–3 d. Acinetobacter junii (ATCC 17908) was grown under aerobic conditions at 26°C for 1–2 d using Nutrient Agar plates (Edge Biologicals). Single-use working stocks of all bacteria were prepared using DPBS and long-term stocks were prepared using 10% glycerol. All bacterial stocks were enumerated using the BD Cell Viability Kit (BD Bioscience) and stored at −80°C until use.
3
Evaluating Murine T-ALL Cell Lines
4
Antibiotic Resistance Evaluation by Flow Cytometry
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This experiment used the PA01:Rms149 resistant strain with no fluorescent label, to avoid interfering with the signal from the stains. We inoculated streptomycin treatment cultures (six replicates per concentration) with 103-fold diluted overnight culture, as in the standard MIC assay. After 7 h of treatment, we diluted test cultures 100-fold and stained with thiazole orange and propidium iodide (BD Cell Viability Kit; product no. 349483). In parallel, we diluted and stained media and heat-killed cultures as controls. We sampled 50 μL per diluted culture using flow cytometry (BD Accuri C6 Flow Cytometer with fast fluidics, discarding events with forward scatter FSC-H < 10,000 or side scatter SSC-H < 8,000). The staining and flow cytometry steps were carried out in groups containing one replicate per concentration plus controls, to avoid potentially toxic effects of stain exposure over prolonged times (SI Appendix, Text, section 7 ). To better discriminate cells from background in the flow cytometry data, we first gated on events according to forward and side scatter before defining clusters of dead (membrane-compromised) and intact cells based on fluorescence; see SI Appendix, Text, section 7 and Fig. S6 , for details.
5
Cellular Uptake of Titanium Dioxide Nanoparticles
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To avoid artifacts associated with dye modification of the size of NPs, we used original NPs not exposed to dye to assess cellular uptake by HepG2 cells and determine the percentage of cells containing NPs.[21 ] Briefly, 2 ml of a HepG2 cell suspension at a density of 5 × 105 cells/well was seeded in a six-well plate and cultured for 24 h. The attached cells were exposed to TiO2 and TiO2-PEG NPs at 0, 100, 200, 400, 600, and 800 μg ml–1. Ultrasonic processing of the NP suspensions for 30 min before and after dilution was employed to ensure adequate dispersion. After 24 h of incubation, cells were trypsinized and passed through a nylon mesh (Cell Strainer Snap Cap) after washing twice with PBS to remove excess NPs. The cells were then collected by centrifugation and suspended in 1 ml of PBS with 6% HFBS. Subsequently, the cells were stained with 1 μl of 42 μM PI (dead cells) and 2 μl of 4.3 mM thiazole orange (all cells) (BD Cell Viability Kit, BD Biosciences, Becton, Dickinson and Co., San Jose, CA, USA). Finally, stained cells were detected using an SP6800 Spectral Analyzer (Sony Biotechnology Inc., Tokyo, Japan). Cell granularity was assessed using side-scattering (SSC) light, and cell size was assessed using forward scattering light.
6
Apoptosis Analysis in CTCL Cell Lines
7
Chitosan Fungal Beads for Biomedical Applications
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To prepare chitosan fungal beads, the washed fungal biomass of S. hirsutum (600 mg dry weight) was homogenized in a sterile blender for 2 min and then centrifuged at 4100 rpm for 10 min to obtain a precipitate. The precipitate was mixed with 50 mL of 2% chitosan (low molecular weight: 50–190 kDa; deacetylation degree: >75%) and encapsulated using a B-390 encapsulator (BÜCHI Labortechnik AG, Flawil, Switzerland) equipped with a concentric nozzle of 1 mm and operated under a vibration frequency of 120 Hz and air pressure of 72 mBar. The dispersed droplets were hardened by treating them with 1% (w/v) sodium tripolyphosphate while stirring magnetically for 24 h to form chitosan-immobilized beads. Chitosan beads were produced under the same conditions but without including fungal biomass. The average size of the resulting chitosan fungal beads was determined using ImageJ software, and their morphology was characterized using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) with a BD Cell Viability Kit to evaluate the structure of the beads.
8
C. difficile Strain Preparation and Quantification
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C. difficile strain VPI 10463 (ATCC, Manassas Virginia) was used for all experiments. Frozen stocks were plated onto TCCFA agar plates (TekNova) and incubated overnight in an anaerobic chamber (Coy, Grass Lake, Michigan). Single colonies were picked and inoculated into BHI Media (Difco) and grown overnight in anaerobic conditions. Cell quantities were quantified with flow cytometry using the BD Cell Viability Kit with BD Liquid Counting Beads (BD Biosciences). Cultures were then centrifuged at 3000 g for 15 min and washed with sterile PBS three times before dilution into sterile water to a final concentration of 9 × 105 cfu/mL.
9
Pluripotent Stem Cell Surface Markers
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Cells were suspended in PBS containing 0.5% bovine serum albumin and 2 mM EDTA and incubated with R-Phycoerythirin-conjugated anti-human SSEA-4 monoclonal antibody (2 µg/106 cells, clone MC-813-70; BioLegend), and Alexa Fluor 488-conjugated anti-human Tra-1-60-R monoclonal antibody (2 µg/106 cells, clone TRA-1-60-R; BioLegend). Cells were resuspended using the BD Cell Viability kit (2 µg/106 cells, BD Bioscience, Franklin Lakes, NJ). The surface expression of target proteins in living cells was analyzed on an LSRFortessa X-20 (BD Bioscience). FCS files were obtained using Diva software and re-analyzed using FlowJo software (BD Bioscience).
10
Cultivation of Anaerobic and Aerobic Bacteria
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