A total of 500,000 cells were plated in 10-cm plates and incubated overnight to allow cells to adhere. The following day, cells were washed three times with PBS and 10 ml of treatment medium with 10% dialysed FBS was added for 96 h. Cells were collected and stained with Live-or-Dye viability dye (Biotium) according to the manufacturer’s instructions. Samples were then passed through a 0.35-µm filter into flow cytometry tubes (Falcon) and run on a BD FACSCanto II Cell Analyzer with 10,000 events recorded for each sample.
Flow cytometry tube
Manufactured by BD
Sourced in United States, United Kingdom
Flow cytometry tubes are specialized containers designed for use in flow cytometry analysis. They are constructed to hold samples for introduction into a flow cytometer, an instrument that measures and analyzes the physical and chemical characteristics of cells or particles suspended in a fluid as they pass through a beam of light.
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Lab products found in correlation
Facscanto 2 cell analyzer, by BD (2 mentions)
Saponin, by Merck Group (2 mentions)
Facsdiva software, by BD (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Tryple, by Thermo Fisher Scientific (2 mentions)
Facsaria 2, by BD (2 mentions)
Cellquest pro analysis software, by BD (2 mentions)
Propidium iodide, by BD (2 mentions)
Annexin 5, by BD (2 mentions)
Click it edu pacific blue kit, by Thermo Fisher Scientific (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Click it edu flow cytometry kit, by Thermo Fisher Scientific (1 mentions)
Goat anti glucagon, by Santa Cruz Biotechnology (1 mentions)
Mouse anti c peptide, by Abcam (1 mentions)
Goat anti sox9, by R&D Systems (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Alexa fluor 488, by BD (1 mentions)
20 protocols using flow cytometry tube
1
Cell Viability Assay by Flow Cytometry
2
Cell Proliferation Assay by Flow Cytometry
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In total, 500,000 cells were plated in 10-cm plates and incubated overnight to allow cells to adhere. The following day, cells were washed three times with PBS and 10 ml of treatment medium with 10% dialysed FBS was added for the desired amount of time. EdU was spiked into cell culture plates at 10 µM for exactly 30 min before fixing. To fix cells, each plate was trypsinized, pelleted and washed twice with PBS. Cells were resuspended in 500 µl ice-cold PBS, and 5 ml ice-cold ethanol was added dropwise to each sample while vortexing in order to obtain a single-cell suspension. Fixed cells were stored at 4 °C until being processed by flow cytometry (no longer than 4 days).
Cells were stained for EdU using the Click-iT EdU Pacific Blue kit (Invitrogen) according to manufacturer instructions. After staining for 30 minutes, cells were stained with propidium iodide. Cells were pelleted and washed with 1% BSA in PBS, then resuspended in 800 µl 1% BSA in PBS. Then, 200 µl propidium iodide/RNAseA staining solution was added to each sample and cells were stained for at least 45 min at 4 °C protected from light. Samples were then passed through a 0.35-µm filter into flow cytometry tubes (Falcon). Samples were run on a BD FACSCanto II Cell Analyzer, and 10,000 events were recorded for each sample. BD FACSDiva software was used to collect data. FlowJo software was used to analyse the data.
Cells were stained for EdU using the Click-iT EdU Pacific Blue kit (Invitrogen) according to manufacturer instructions. After staining for 30 minutes, cells were stained with propidium iodide. Cells were pelleted and washed with 1% BSA in PBS, then resuspended in 800 µl 1% BSA in PBS. Then, 200 µl propidium iodide/RNAseA staining solution was added to each sample and cells were stained for at least 45 min at 4 °C protected from light. Samples were then passed through a 0.35-µm filter into flow cytometry tubes (Falcon). Samples were run on a BD FACSCanto II Cell Analyzer, and 10,000 events were recorded for each sample. BD FACSDiva software was used to collect data. FlowJo software was used to analyse the data.
3
Flow Cytometric Analysis of Transfected Cells
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To perform analysis by flow cytometry, cells were detached using trypsin and transferred to flow cytometry tubes (BD Falcon, Radnor, PA, USA). Next, the cell suspensions were centrifuged at 300× g for 5 min (Bio-Rad DiaCent-12, DieMed GmbH, Cressier, Switzerland) and resuspended in flow buffer (DPBS-, 0.1% Sodium Azide, 1% Bovine Serum Albumine). Finally, samples were vortexed at 2200 rpm (YellowLine TTS2, IKA works, Wilmington, DC, USA). Flow cytometry was performed on 10,000 events per sample (CytoFLEXTM Flow Cytometer, Beckman Coulter, Krefeld, Germany) or for a total duration of 120 s. gWIZ GFP fluorescence was detected with 525/40 nm bandpass filter after 488 nm excitation. DAPI fluorescence was detected with a 450/45 nm bandpass filter after 405 nm excitation. FlowJo software (version, Treestar Inc., Ashland, OR, USA) was used to perform the analysis.
4
Flow Cytometric Profiling of Cell Populations
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Differentiated cell clusters were dispersed into a single-cell suspension with TrypLE (Life Technologies) at RT, fixed with 4% paraformaldehyde at 4 °C, washed three times in PBS + 0.2% bovine serum albumin (Millipore) + 0.1% saponin (Sigma), blocked for 30 min at 4 °C in PBS + 5% donkey serum + 0.1% saponin, incubated with primary antibodies overnight at 4 °C, washed, and incubated with secondary antibodies for 1 h at RT. Stained, fixed cells were filtered through a 40- μm nylon mesh into flow cytometry tubes (BD Falcon) and were analyzed for relevant stage-specific marker expression using LSR II flow cytometers (BD Biosciences) and FlowJo for the data analysis. Samples stained with secondary antibodies only were used to gate on cells that are negative and accurately identified cells expressing the markers included in the analysis. For shRNA-infected samples, only infected GFP + cells were included in the analysis (approximately in 30–40% of all cells). To quantify incorporation of EdU and estimate levels of proliferation, cells pulsed with 10 μM EdU for 4 h were dissociated as described above unless stated otherwise. The Click-IT EdU flow cytometry kit (Invitrogen) was used to detect EdU incorporation following the manufacturer’s protocol with minor modifications.
5
Flow Cytometry Analysis of hPSC-Derived Cells
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Differentiated hPSC-MPCs and hPSC-β cells were dissociated using TrypLE (Thermo Fisher Scientific, USA). Cells were fixed with 4% paraformaldehyde at 4 °C for 1 hour or overnight. They were later washed three times in PBS containing 0.2% bovine serum albumin (BSA) (Sigma, USA) and 0.1% saponin (Sigma, USA), blocked for 30 min at 4 °C in PBS containing 5% donkey serum and 0.1% saponin and then they were incubated with primary antibodies overnight at 4 °C. The primary antibodies were mouse anti-NKX6.1 (1:100; DSHB), guinea pig anti-PDX1 (1:100; Abcam), rabbit anti-Chromogranin A (CHGA) (1:1000; Abcam), goat anti-SOX9 (1:300; R&D Systems) and mouse anti- C-Peptide (1:100; Abcam), goat anti-Glucagon (1:250; SantaCruz). Cells were washed with PBS containing 0.2% BSA and 0.1% saponin and incubated with secondary antibodies in PBS for 1 h at room temperature. Stained cells were filtered through a 40-μm nylon mesh into flow cytometry tubes (BD Falcon) and were analyzed with BD Accuri™ C6 flow cytometer (BD Biosciences, USA) and FlowJo for data analysis.
6
Cardiac Differentiation Marker Analysis
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Cells at differentiation day 6, 8, and 10 were dissociated into single cells by Trypsin-EDTA and transferred to flow cytometry tubes (BD Biosciences, San Jose, CA, USA) followed by fixation and permeabilization using the BD Cytofix/Cytoperm Kit (BD Biosciences, San Jose, CA, USA) with addition of 1:200 mouse anti-Tnnt2 antibody (Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes at room temperature. Secondary antibody staining was performed in 1:1000 Alexa Fluor 488 goat anti-mouse IgG1 for 30 minutes. Cells were analyzed using the FACSCanto II and the FACSDiva software (BD Biosciences, San Jose, CA, USA). Data were analyzed using the FlowJo 10.1 software (FlowJo, LLC, Ashland, OR, USA).
7
Pluripotency Assessment of Human iPSCs
8
Flow Cytometry Analysis of MSCs
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When MSCs reached 80–90% confluency, cells were washed with 1 × PBS, dissected to single cells, and finally resuspended in 1.2 ml of 1 × PBS containing 3% FBS. Cells were aliquoted equally to six different tubes and stained with fluorescent antibodies (BD 562,245) for 30 min protected from light. Cells were washed twice with 1 × PBS and resuspended with 150 µl 1 × PBS containing 3% FBS and transferred to Flow Cytometry Tubes for BD FACSAria SORP analysis. Data were analyzed with FlowJo 10.7.
9
Cardiac Differentiation Efficiency Assessment
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For assessment of cardiac differentiation efficiency, cells on day 15 of differentiation were dissociated using Cell Dissociation Solution (Cellapy) for 25 minutes at 37°C and transferred to flow cytometry tubes (BD Biosciences). Cells were then fixed with 4% paraformaldehyde (PFA) for 10 minutes, permeabilized with 0.1% saponin for 20 minutes, and stained using 1:100 mouse monoclonal IgG1 TNNT2 (Santa Cruz) for 30 minutes at RT. Isotype control Alexa Fluor 594 mouse IgG (Life Technology) was used to establish gating. Cells were then analysed using a flow cytometre.
10
Cardiac Differentiation and Flow Cytometry Analysis
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WTSIi020-A-CMs and UKKi025-A-CMs were harvested on day 0 and 15 to evaluate the efficacy of the differentiation protocol (CDM3) in producing hiPSC-CMs. The dissociated cells were directly transferred to flow cytometry tubes (BD Biosciences) and stained with a live/dead discriminator using 1:1000 Zombie Aqua Dye (BioLegend, #423101). Samples were fixed with 4% PFA for 15 min and stored in FACs buffer, containing 2% FBS, 5mM EDTA, 0.01% Sodium Azide in PBS, at 4 °C for later use. Before flow cytometry analysis, samples were permeabilized using a blocking buffer containing 20% FBS, 3% BSA, 0.2% Saponin in PBS for 10 min at RT. They were then stained using an antibody solution containing 1% BSA and 0.1% Saponin in PBS, 1:600 mouse monoclonal IgG1 TNNT2 (cardiac troponin T, clone 1G1, #MA5-17192) and rabbit monoclonal IgG OCT4 (Octamer-binding transcription factor 4, clone T.631.9, #MA5-14845) primary antibodies (ThermoFisher Scientific, USA) for 1 h in the dark at RT. Secondary antibody staining was performed with 1:600 dilutions of Alexa Fluor 488 and Alexa Fluor 568 secondary antibodies (Life Technologies) for 30 min at RT. Samples were analyzed using LSRFortessa (BD Biosciences) and FACSDiva Software. The resulting data was analyzed using FlowJo v10 (TreeStar).
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