For supernatant transfer assays to assess virion production, BAC16-containing iSLK cells were plated at a density of 1x106 cells in 10 cm dishes and treated with 1 ug/mL doxycycline and 1 mM sodium butyrate the following day. 72 h post induction, the supernatant was filtered through a 0.45 uM syringe filter and 2 ml of the supernatant was mixed with 1x106 freshly trypsinized HEK293T cells in a 6 well plate and centrifuged at 1,500xg for 2 h at 37°C. The following day, cells were trypsinized, fixed in 4% paraformaldehyde and the percentage of cells expressing GFP was determined by flow cytometry (BD Csampler, BD Biosciences).
Csampler
Manufactured by BD
Sourced in United States
The BD CSampler™ is a laboratory equipment designed for collecting and storing biological samples. It facilitates the automated processing and handling of samples, ensuring consistent and reliable sample collection.
Automatically generated - may contain errors
Lab products found in correlation
Accuri c6, by BD (3 mentions)
Accuri c6 flow cytometer, by BD (3 mentions)
Bodipy 505 515, by Thermo Fisher Scientific (2 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (1 mentions)
Incucyte s3 live cell analysis system, by Sartorius (1 mentions)
B8631, by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Syto9, by Thermo Fisher Scientific (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Fcs express 4 flow research edition software, by De Novo Software (1 mentions)
10 protocols using csampler
1
Supernatant Transfer Assay for Virion Production
2
Quantitative Data Analysis Protocol
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The results were accumulated, processed for graphing, and data analysis was done with Origin Pro 8 SRO (v8.0724) and BD CSampler™ software. All experiments were done in triplicate to monitor for the reproducibility of the results, and all data is expressed as the mean ± standard deviation. Difference between groups were determined using the one-tailed student t-test.
3
Oxidative Stress Response in K562 Cells
4
Cultivation and Analysis of P. soloecismus
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P. soloecismus was grown in f/2 media containing half the recipe nitrogen and using Instant Ocean sea salt (Blacksburg, VA) at 38 g/L [31 ,32 (link)]. Cultures were grown at room temperature on a 16 hour light/8 hour dark cycle and mixed by stirring. PH was maintained at 8.25 with on-demand CO2 injection when the pH increased above the set-point. Cells were collected and stored at 4 °C prior to analysis.
Stained populations of cells were incubated with 22.6 µM BODIPY 505/515 (Thermo Fisher Scientific) with 2.8% DMSO in media for 30 minutes at room temperature prior to analysis. Analysis was conducted using a BD Accuri™ C6 flow cytometer with BD CSampler™ (BD Biosciences). Unstained samples were collected with a set volume of 10 µl on a high flow rate (66 µl/min), for stained samples 10,000 events were collected on a low flow rate (14 l/min). Data was exported in .csv format for subsequent analysis.
Stained populations of cells were incubated with 22.6 µM BODIPY 505/515 (Thermo Fisher Scientific) with 2.8% DMSO in media for 30 minutes at room temperature prior to analysis. Analysis was conducted using a BD Accuri™ C6 flow cytometer with BD CSampler™ (BD Biosciences). Unstained samples were collected with a set volume of 10 µl on a high flow rate (66 µl/min), for stained samples 10,000 events were collected on a low flow rate (14 l/min). Data was exported in .csv format for subsequent analysis.
5
Cytotoxic Effects of Peptide Treatments
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Cells were seeded into 96-well plates at a density of 5 × 105 cells/mL and incubated for 24 h in a medium containing 10% FBS at 37 °C in 5% CO2. Then, cells were treated with a medium containing 5 µM of peptides HB43, mut3, and 20 µM of mut2 and incubated for 12 h at 37 °C. Cells were washed with PBS and a hypotonic propidium iodide citrate stain (50 μg/mL in 0.1% trisodium citrate dihydride) containing 0.3 μL/mL of Nonidet P-40 was added. Finally, the adherent cells were harvested by scraping vigorously with a tip of a pipette to dislodge any cytoplasmic remnants from the lysed cells and kept at 4 °C until analysis. The stained cells were analyzed using a BD Accuri™ C6 flow cytometer integrated with a BD CSampler (BD Biosciences, San Jose, CA, USA).
6
Bile Salts Survival Assays
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Porcine bile (B8631; Sigma) and bovine bile (B3883; Fluka) were diluted in MRS (Merck) to a final concentration of 0.5% (w/v) and 1% (w/v) bile salts, respectively. Low pH MRS was prepared by adding 1 M HCl to MRS, lowering the pH to pH 2. Freeze-dried cell samples were rehydrated for 20 min with 1 mL saline solution, vortexed and diluted 100 times in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) to reach a target concentration. One mL samples were spun down for 2 min at 16,100×g. The cells were then resuspended in 1 mL MRS-bile salts or low pH-MRS and vortexed, immediately after which 100 µL samples were taken for flow cytometry analysis. The remaining samples with MRS-bile salt or low pH-MRS were then incubated at 37 °C and new samples for survivability assays were taken every 30 min. The survival of the cells was checked with a flow cytometer (BD Accuri C6 plus flow cytometer with a BD C sampler (BD Biosciences, Franklin Lakes, NJ, USA) using a protocol described below.
7
Immunophenotyping of Human Mesenchymal Stem Cells
8
Cultivation and Analysis of P. soloecismus
9
Cell Viability Quantification via Flow Cytometry
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The extracted foulant mixture was first passed through a 40 µm filter. The cell viability was quantified using a flow cytometer (BD, USA). Specifically, 1 mL of sample was stained with 1 µL of SYTO9 and PI each (Molecular Probes, USA). The sample was transferred to a flat-bottom well-plate for flow cytometry analysis (with an unstained sample as a control). Proprietary software (CSampler, BD, USA) was used to process the results. Counts in a defined region of a density plot were converted to live and dead cell counts.
10
Mitochondrial Membrane Potential Assay
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Cells were pulsed with 5 μM MitoSOX Red or 1 nM TMRE (tetramethylrhodamine, ethyl ester) and 10 nM TO-PRO-3 (Thermo Fisher Scientific). A minimum of 10,000 live events were collected using an Accuri C6 (BD Biosciences) and analyzed using CSampler (BD Biosciences) and FCS Express4 Flow Research edition software (DeNovo). For MitoSOX Red and TMRE experiments, dyes were excited at 488 nm and emitted fluorescence was detected using the FL2 (585/40 nm) and the FL3 (670 LP) filters, respectively.
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