Transduction of lentiviral vector into Ramos B cells was performed with 8 μg/ml protamine sulfate (Sigma). CRISPR-mediated knockout cells were enriched by culturing in B cell medium supplemented with 1–2 μg/ml puromycin (Invitrogen). CD19 knockout Ramos B cells were purified using a FACSAria II (BD Bioscience). For this, cells were washed and then stained with anti-CD19 APC (clone SJ25-C1; BD Bioscience) in phosphate buffered saline (PBS; Fresenius Kabi) supplemented with 0.1% bovine serum albumin (BSA; Sigma). The NCK1/2 double-knockout cell line was obtained by single cell sorting using a FACSAria II (BD Bioscience). After clonal expansion, cells were screened for complete knockout using an immunoblot assay (as described below). Ramos B cells that stably expressed Lifeact-GFP or RAC1 biosensor were sorted by flow cytometry-based sorting using a FACSAria II (BD Bioscience).
Facsaria 2
Manufactured by BD
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The FACSAria II is a high-performance cell sorter produced by BD. It is designed for precision cell sorting and analysis. The system utilizes flow cytometry technology to rapidly identify and separate different cell populations within a sample.
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Lab products found in correlation
Facsdiva software, by BD (152 mentions)
Lsrfortessa, by BD (129 mentions)
Facscanto 2, by BD (113 mentions)
Facscalibur, by BD (67 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (60 mentions)
Facsdiva, by BD (58 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (55 mentions)
Lsr 2, by BD (52 mentions)
Facsaria fusion, by BD (48 mentions)
Propidium iodide, by Merck Group (43 mentions)
Facsverse, by BD (39 mentions)
Dnase 1, by Roche (37 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (36 mentions)
Facsaria 3, by BD (35 mentions)
Propidium iodide, by Thermo Fisher Scientific (35 mentions)
Cytofix cytoperm kit, by BD (34 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (34 mentions)
Fortessa, by BD (31 mentions)
Hoechst 33342, by Thermo Fisher Scientific (31 mentions)
Polybrene, by Merck Group (30 mentions)
4 727 protocols using facsaria 2
1
Lentiviral Transduction and CRISPR Knockout in Ramos B Cells
2
Isolation and Purification of Murine Thymocyte and Stromal Cell Subsets
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To isolate DP and SP subsets for RT-PCR analysis, thymocytes from a 1-mo-old C57BL/6J mouse were immunostained with anti-CD3, CD4, CD8, CD69, Gr1, CD11b, NK1.1, CD25, and PI. 106 cells from each indicated thymocyte subset were sorted to >95% purity on a FACSAria II (BD). To isolate DN thymocyte subsets, thymocytes from 6 C57BL/6J mice were pooled and DP and CD8 SP thymocytes were depleted by immunostaining with anti-CD8 antibody, followed by magnetic depletion with anti–rat IgG DynaBeads (Life Technologies) according to the manufacturer’s guidelines. The remaining cells were stained with fluorescently conjugated antibodies to CD4, CD8, CD25, CD44, and cKit. 5 × 104 DN1-4 cells were sorted to >95% purity on a FACSAria II (BD).
Thymic stromal cell subsets were sorted as previously described (Ki et al., 2014 (link)). In brief, thymi from 1-mo-old male C57BL/6J mice were digested with Collagenase D (Roche), followed by Collagenase/Dispase (Roche) in the presence of DNase I, as described previously (Gray et al., 2008 (link)). Cells were immunostained with FITC-conjugated Ulex europaeus agglutinin I (UEA-1; Vector Laboratories) and the following fluorochrome-conjugated antibodies: EpCAM, TER-119, CD11c, CD31, Sirpα, B220, I-A/I-E, CD80, CD45, and Ly-51. Stromal subsets were FACS purified by double sorting to >95% purity on a FACSAria II (BD).
Thymic stromal cell subsets were sorted as previously described (Ki et al., 2014 (link)). In brief, thymi from 1-mo-old male C57BL/6J mice were digested with Collagenase D (Roche), followed by Collagenase/Dispase (Roche) in the presence of DNase I, as described previously (Gray et al., 2008 (link)). Cells were immunostained with FITC-conjugated Ulex europaeus agglutinin I (UEA-1; Vector Laboratories) and the following fluorochrome-conjugated antibodies: EpCAM, TER-119, CD11c, CD31, Sirpα, B220, I-A/I-E, CD80, CD45, and Ly-51. Stromal subsets were FACS purified by double sorting to >95% purity on a FACSAria II (BD).
3
Quantification of Serum Cytokine Levels
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The serum concentrations of IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10 and IL-17A were determined using CBA [34 (link)], according to the manufacturer’s protocol (BD Biosciences) with minor modifications using a flow cytometer (BD FACSAriaTM II, Becton Dickinson, USA). The concentrations of serum cytokines were quantified using the Cell Quest Pro according to routine procedures and CBA software (Becton Dickinson) on a FACSAria II.
4
Multiparameter Flow Cytometry Analysis
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Immunophenotype analysis was performed on either a LSRFortessa™ cell analyzer or FACSAria II and cell sorting on a FACSAria II or FACSAria III sorter (Becton Dickinson Biosciences). Antibodies used for immunophenotype determination and subpopulation sorting are described in the Online Supplementary Methods.
5
Quantification of α6 Integrin Expression in Hypoxia
6
Thymus Cell Proliferation and Apoptosis Analysis
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For cell proliferation analysis, thymus cells were first stained for the indicated cell surface markers. After fixation and permeabilization (BD Biosciences), the cells were stained with FITC-conjugated anti-Ki-67 and 7-AAD (eBiosciences, San Diego, CA). Data were acquired by a FACS Aria II (Becton Dickson) and analyzed using FlowJo software.
For cell apoptosis analysis, thymus cells were first stained for the indicated surface markers. After washing with buffer, the cells were then stained with anti-Annexin V and 7-AAD (eBiosciences). Data were acquired by a FACS Aria II (Becton Dickson) and analyzed using FlowJo software.
For cell apoptosis analysis, thymus cells were first stained for the indicated surface markers. After washing with buffer, the cells were then stained with anti-Annexin V and 7-AAD (eBiosciences). Data were acquired by a FACS Aria II (Becton Dickson) and analyzed using FlowJo software.
7
Isolating Intestinal Stem Cell Populations
8
CRISPR-Edited Cell Line Generation
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Transfected Y75 cells sorted on a Becton Dickinson FACS Aria II cell sorter were first seeded into a 24-well plate at the density of 5 cells/well. 7 days after the seeding single or mixed colonies would appear. 14 days after the seeding cells were ready to make replica plates for genotyping and human specific FMR1 qPCR. Genotyping positive colonies were further seeded into a 96-well plates at the density of 0.5 cell/well to isolate morphologically single colonies. After confirming purified single colonies by genotyping, colonies were viewed as pure CRISPR cut clonal lines.
Transfected iPS cells sorted on a Becton Dickinson FACS Aria II cell sorter were first seeded onto a MEF layered 24-well plate at the density of 100 cells/well. The MEF coating density was 4 x 104 cells/cm2. iPS cell aggregates appear between d7 and d10 post sorting. At this time point, pour a new layer of MEF to coat the plate again to substitute the old MEF layer. Continue feeding the cells until iPSC colonies were big enough for toothpick PCR based CRISPR deletion genotyping. Deletion positive colonies were transferred to the matrigel plate and cultured as pure CRISPR cut clonal lines.
Transfected iPS cells sorted on a Becton Dickinson FACS Aria II cell sorter were first seeded onto a MEF layered 24-well plate at the density of 100 cells/well. The MEF coating density was 4 x 104 cells/cm2. iPS cell aggregates appear between d7 and d10 post sorting. At this time point, pour a new layer of MEF to coat the plate again to substitute the old MEF layer. Continue feeding the cells until iPSC colonies were big enough for toothpick PCR based CRISPR deletion genotyping. Deletion positive colonies were transferred to the matrigel plate and cultured as pure CRISPR cut clonal lines.
9
Analysis of T-Cell Receptor Clonality
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Peripheral blood samples were stained for lymphocyte clonality analysis with anti-CD3 (SK7), anti-CD4 (SK3), and anti-CD8 (SK-1) (Becton Dickinson) and a panel of T-cell receptor β variable chain (TCR Vβ) antibodies (IOTest Beta Mark TCR V kit, Beckman Coulter Immunotech, cat. no IM3497), which recognize approximately 70% to 80% of the human TCR β V regions. After staining, red blood cells were lysed with BD FACS Lysing Solution (Becton Dickinson Biosciences) and re-suspended to phosphate-buffered saline (PBS) with 2 mM EDTA. Samples were acquired with FACSAria II or FACS Verse (Becton Dickinson) and analyzed with FlowJo software (Becton Dickinson).
Cell sorting was performed either from fresh or from cryopreserved PBMCs using FACSAria II (Becton Dickinson) or Sony SH800 (Sony Biotechnology Inc.). For sorting, MNCs were stained with anti-CD3 (SK7), anti-CD4 (SK3), anti-CD8 (SK-1), and the appropriate anti-Vβ antibody from the IOTest Beta Mark TCR V kit. Cell fractions’ purities were controlled with flow cytometry, and the purities of all sorted fractions were nearly 100%.
Cell sorting was performed either from fresh or from cryopreserved PBMCs using FACSAria II (Becton Dickinson) or Sony SH800 (Sony Biotechnology Inc.). For sorting, MNCs were stained with anti-CD3 (SK7), anti-CD4 (SK3), anti-CD8 (SK-1), and the appropriate anti-Vβ antibody from the IOTest Beta Mark TCR V kit. Cell fractions’ purities were controlled with flow cytometry, and the purities of all sorted fractions were nearly 100%.
10
Cell Viability, Proliferation, and Apoptosis Assays
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The CCK-8 detection kit (Dojindo, Shanghai, China) was used to measure cell viability. Cells were seeded in a 96-well (100 μl per well) plates at a density of 5 × 104/ml. CCK-8 solution (10 μl per well) was added and the plate was incubated at 37 °C for 2 h. The viable cells were counted by absorbance measurements with a monochromator microplate reader at a wavelength of 450 nm.
BrdU incorporation and cell proliferation analysis were detected using BD Pharmingen™ BrdU Flow Kit (BD, San Diego, CA, USA). Cells were plated into 6-well plates and transfected with siRNA for 24 h, added BrdU (1 mM/mL) for 12 h and then harvested. Cells were stained with anti-BrdU and 7-AAD according to the manufacturer’s instructions. Cell cycle distribution was determined by flow cytometry (FACSAriaTM II, Becton Dickinson, Mountain View, CA, USA).
The Annexin V-PE assay kit (BD) was utilized to analyze cell apoptosis in GBM cells. After transfected with siRNA for 24 h, cells were harvested and resuspended in 100 ul binding buffer at a density of 1 × 106 cells/ml. PE-conjugated Annexin V and 7-AAD reagent staining were performed in concentrations and time recommended by the manufacturer. Stained cells were analyzed by flow cytometry (FACSAria II, BD).
BrdU incorporation and cell proliferation analysis were detected using BD Pharmingen™ BrdU Flow Kit (BD, San Diego, CA, USA). Cells were plated into 6-well plates and transfected with siRNA for 24 h, added BrdU (1 mM/mL) for 12 h and then harvested. Cells were stained with anti-BrdU and 7-AAD according to the manufacturer’s instructions. Cell cycle distribution was determined by flow cytometry (FACSAriaTM II, Becton Dickinson, Mountain View, CA, USA).
The Annexin V-PE assay kit (BD) was utilized to analyze cell apoptosis in GBM cells. After transfected with siRNA for 24 h, cells were harvested and resuspended in 100 ul binding buffer at a density of 1 × 106 cells/ml. PE-conjugated Annexin V and 7-AAD reagent staining were performed in concentrations and time recommended by the manufacturer. Stained cells were analyzed by flow cytometry (FACSAria II, BD).
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