The differentiated cells in stage 3 (day 12) were treated with 0.25% trypsin/ethylenediaminetetraacetic acid for 5 min at 37°C. After dissociation, the cells were fixed and permeabilized using BD cytofix/cytoperm™ fixation/permeabilization solution (BD Biosciences). The primary antibody was guinea pig anti‐INS (1:500), and the secondary antibody was Alexa Fluor® 647 AffiniPure Donkey Anti‐Guinea Pig IgG (H+L; Jackson ImmunoResearch, West Grove, PA, USA). The cells were analyzed and sorted by FACS Aria II (Becton, Dickson and Co., Franklin Lakes, NJ, USA).
Cytofix cytoperm fixation permeabilization solution
Manufactured by BD
Sourced in United States
Cytofix/Cytoperm Fixation/Permeabilization solution is a laboratory reagent used to fix and permeabilize cells. It is designed to enable intracellular staining and analysis of cellular proteins and other intracellular components.
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Lab products found in correlation
Perm wash buffer, by BD (7 mentions)
Ionomycin, by Merck Group (3 mentions)
Perm wash, by BD (2 mentions)
Accuri c6, by BD (2 mentions)
Lsrfortessa cell analyzer, by BD (2 mentions)
Cytoflex, by Beckman Coulter (2 mentions)
Trustain fcx anti mouse cd16 32, by BioLegend (2 mentions)
Brefeldin a, by Thermo Fisher Scientific (2 mentions)
Monensin, by Thermo Fisher Scientific (2 mentions)
Lsr 2, by BD (2 mentions)
Brilliant violet 711 anti mouse cd8a antibody, by BioLegend (2 mentions)
Percp cyanine5.5 anti mouse cd14 antibody, by BioLegend (2 mentions)
Pe anti mouse nk 1.1 antibody, by BioLegend (2 mentions)
Apc cyanine7 anti mouse ifn γ antibody, by BioLegend (2 mentions)
Live dead fixable violet stain kit, by Thermo Fisher Scientific (2 mentions)
Fitc anti mouse cd3 antibody, by BioLegend (2 mentions)
Apc anti mouse cd4 antibody, by BioLegend (2 mentions)
Lsrfortessa, by BD (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
34 protocols using cytofix cytoperm fixation permeabilization solution
1
Insulin-Producing Cell Isolation
2
Quantifying DNA Damage in Irradiated Cells
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The DNA damage in the irradiated PNT1A cells was assessed with the Apoptosis, DNA Damage and Cell Proliferation Kit (BD Biosciences, Franklin Lakes, NJ, USA). The cells were kept at room temperature until collection. The cells were collected using the Accutase cell detachment solution (Biowest, Nuaillé, France). Cells were first washed with 1× BD Perm/Wash™ Buffer (BD Biosciences, Franklin Lakes, NJ, USA), and 5 × 105 cells were fixed using the BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution (BD Biosciences, Franklin Lakes, NJ, USA). The cells were fixed two hours after irradiation. Next, the cells were stained with an anti-γH2AX antibody (mouse, Alexa Fluor® 647 conjugated). The stained cells were analysed using a CytoFLEX flow cytometer (Beckman Coulter Life Sciences, IN, USA) and FlowJo V10 software (FlowJo LCC, Ashland, OR, USA). For the fluorescence signal quantification, the percentage of positive cells was evaluated.
3
Multiparametric Flow Cytometry Analysis of Dissociated Cells
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Dissociated cells labeled as detailed above followed by 1 h fixation at 4°C using the BD Cytofix/Cytoperm® fixation/permeabilization solution (BD Biosciences) as in Morales-Tirado et al. (2004 (link), 2010 (link), 2011 (link)) and Kasow et al. (2011 (link)). Cells were incubated for 1 h at 4°C in the following antibodies that were diluted in BD Perm/Wash (BD Biosciences) buffer: anti-RNA-Binding Protein With Multiple Splicing (RBPMS; rabbit polyclonal IgG; Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:100 dilution); anti-SNCG (rabbit polyclonal IgG; GeneTex, 1:100 dilution), Brain-Specific Homeobox/POU Domain Protein 3A (BRN3A; goat ployclonal IgG; Santa Cruz Biotechnology; 1:100 dilution); and anti-TUJ1 (mouse monoclonal IgG2a; Covance; 1:100 dilution). The appropriate Alexa Fluor 488 tagged secondary antibodies (1:200 dilution; Invitrogen) were used to allow for data acquisition and analysis. Cells were kept in PBS/1%FBS on ice until the time of analysis. Data acquisition was performed on a BD LSRII Flow Cytometer (BD Biosciences) and analyses were performed using FlowJo vX10.0.6 (Tree Star, Inc., Ashland, OR, USA). To confirm results we performed additional data acquisition using the Miltenyi Biotec MACSQuant® 10 Analyzer (Myltenyi Biotec, San Diego, CA, USA).
4
Multiparametric Analysis of Cell Proliferation
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Cell were fixed and permeablilized with BD Cytofix/Cytoperm™ Fixation/Permeabilization solution after cell surface staining. Ki-67 was performed using the PE-mouse anti-human Ki-67 kit (BD Bioscience). For analysis of BrdU incorporation, bone marrow cells were incubated with 10 μM BrdU (Sigma) at 37°C for an hour. After cell surface staining, BrdU staining were performed using BD APC flow kit.
5
Wound Fluid-Induced Cellular Stress Response
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Cells were stimulated with wound fluids and conditioned medium and analysed at 9 time points: 30 min and 1, 2, 4, 8, 24, 48, 72 and 96 h after addition of fluids. Cells were then collected using Accutase (Biowest, France), fixated with BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution (BD Biosciences, NJ, USA) and stained with fluorochrome-conjugated monoclonal antibodies: anti-human active caspase-3 antibody (Alexa Fluor 647 conjugated, rabbit IgG) (BD Biosciences, NJ, USA, Catalogue No. 552933), anti-human cleaved PARP antibody (PE conjugated, mouse IgG1) (BD Biosciences, NJ, USA Catalogue no. 552933) and anti-human γH2AX antibody (Alexa Fluor 488 conjugated, mouse IgG1) (BD Biosciences, NJ, USA Catalogue No. 560445). The stained cells were analysed using BD Accuri C6 (BD Biosciences, NJ, and USA). For quantification of each fluorescence signal, the median fluorescence intensity (MFI) was used. The results were normalized to the MFI of control (untreated) cells for each time point analysed.
6
Apoptosis and Differentiation Analysis of Cells
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Cells were resuspended to a concentration of 1 × 106 cells were plated in triplicate in a 12-well tissue culture plate. For assessment of annexin V staining, cells were washed with PBS and then resuspended in PBS with annexin V-APC (BioLegend) and propidium iodide at a dilution of 1:1000. For intracellular detection of cleaved caspase 3, cells were fixed and permeabilized using the BD Cytofix/Cytoperm Fixation/Permeabilization solution according to the manufacturer’s instructions (BD Biosciences). Cells were then stained using the Alexa Fluor 647-conjugated anti-active caspase-3 (BD Biosciences) at a dilution of 1:50. Cells were incubated for 30 minutes room temperature in the dark, washed, and then analyzed using the BD LSRFortessa cell analyzer. For assessment of differentiation, cells were stained using the anti-human CD14 PE at a dilution of 1:20 (Affymetrix eBiosciences) and anti-human CD66b at a dilution of 1:20 (Affymetrix eBiosciences).
7
Quantifying Apoptosis in LNCaP Cells
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LNCaP cells (un- and treated with different concentrations of leptins) were stained for cPARP with the PE Mouse Anti-Cleaved PARP (Asp214) antibody (562253, BD Biosciences, NJ, USA) according to manufacturer’s instructions. Briefly, 1 × 106 un- and treated cells were fixed and permeabilized with BD Cytofix/Cytoperm Fixation/Permeabilization Solution for 30 min at room temperature. Then, the additional permeabilization and fixation was performed. The fixed cells were washed with BD Perm/Wash Buffer and stained with appropriate antibody (5 μL/test) for 20 min at room temperature. The stained and washed cells were resuspended in 500 μL PBS and analyzed with a flow cytometer (CytoFLEX, Beckman Coulter, CA, USA). Fluorescence intensity in arbitrary units was plotted in histograms and the mean fluorescence intensity was calculated. Data were analyzed using FlowJo software (FlowJo v10; LLC, Ashland, OR, USA).
8
Multiparametric Flow Cytometry Analysis of PBMCs
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Peripheral blood mononuclear cells (PBMCs) were cleared of red blood cells using the red blood cells lysis buffer from Alfa Aesar (Thermo Fisher Scientific, UK) according to the manufacturer’s instructions. Cells were fixed and stained using BD Cytofix/Cytoperm Fixation/Permeabilization Solution (BD, UK) and analysed using a BD Fortessa flow cytometer (BD). Flow cytometry data were analyzed using FlowJo software (Tree Star). PE-Cy7 rat anti-mouse CD11b, APC-eFluor780 rat anti-mouse CD11c, PE rat anti-mouse CD14, Pacific blue rat anti-mouse CD3, eFluor650 rat anti-mouse CD4 and APC rat anti-mouse CD8a were from BD Biosciences (UK). Anti-Flavivirus Group Antigen antibody, clone D1-4G2-4-15 was obtained from Merck (UK). The secondary Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) was from Thermo Fisher Scientific (UK).
9
Multiparametric Analysis of Tumor Immune Cells
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Tumor single-cell suspensions were stained with BD Horizon™ Fixable Viability Stain 700 (FVS700), and surface markers were stained. Then, the cells were permeabilized and fixed with BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution (BD Biosciences, USA) at 4 °C for 20 min. The cells were washed twice with 1× Perm/Wash buffer and stained with fluorescence-conjugated intracellular cytokine antibodies, such as anti-IFNγ-APC, at 4 °C for 30 min. The cells were washed again and resuspended in PBS for flow cytometric analysis.
10
Apoptosis and DNA Damage Assay
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Cells were treated with chemotherapy, alone or in combination with nicotine or cotinine, for 48 h. Cells were then collected and resuspended in 1× Binding Buffer (1 × 106 cells/mL). Cells suspension (100 µL) was mixed with 5 µL antibody against Annexin V and 5 µL PI and incubated for 20 min, according to the manufacturer’s instructions (Invitrogen, Cat. 88800774 Waltham MA, USA). For γH2A.X and cleaved PARP staining, cells were collected, fixed, and permeabilized with BD Cytofix/Cytoperm Fixation/Permeabilization Solution. Finally, cells were incubated with BD Perm/Wash Buffer (50 µL), with anti-H2AX (5 µL/test), and with anti-Cleaved PARP antibodies, followed by Accuri C6 flow cytometry analysis.
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