The Fixation/Permeabilization solution and Perm/WashTM Buffer (BD Biosciences, San Jose, CA, USA) were used. The anti-Sox2 antibody (S9072, Sigma-Aldrich) and the secondary Alexa Fluor® 647-conjugated antibody (Thermo Fisher Scientific, Waltham, MA, USA) were diluted 1:200 and 1:500, respectively. The FACS Canto instrument and the FACS Diva and FlowJo software version 6.1.1 (BD Biosciences) were used for the analysis.
Facscanto instrument
Manufactured by BD
Sourced in United States
The FACSCanto instrument is a flow cytometry system designed for research applications. It is capable of multiparametric analysis of single cells or particles in a fluid stream. The instrument utilizes lasers and detectors to measure and record the physical and fluorescent characteristics of the samples.
Automatically generated - may contain errors
Lab products found in correlation
Facsdiva software, by BD (4 mentions)
Il 17 ebio17b7, by BD (2 mentions)
L calc software, by STEMCELL (2 mentions)
Cd45 apc, by BD (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Perm wash buffer, by BD (1 mentions)
Alexa fluor 647 conjugated antibody, by Thermo Fisher Scientific (1 mentions)
Fixation permeabilization solution, by BD (1 mentions)
Cd4 rm4 5, by BD (1 mentions)
Cd8 53 6, by BD (1 mentions)
Ifn γ xmg1.2, by BD (1 mentions)
Mouse foxp3 buffer set, by BD (1 mentions)
7 amino actinomycin d 7 aad viability staining solution, by BioLegend (1 mentions)
Rbc lysing buffer, by BioLegend (1 mentions)
Facsdiva software v8, by BD (1 mentions)
αcd3 cd2 cd28 t cell activation beads, by Miltenyi Biotec (1 mentions)
Directly conjugated anti human monoclonal antibodies, by BioLegend (1 mentions)
Phycoerythrin cyanine7 anti ifn γ xmg1.2, by Thermo Fisher Scientific (1 mentions)
Aqua live dead fixable kit, by Thermo Fisher Scientific (1 mentions)
Fc block 2.4g2, by BD (1 mentions)
37 protocols using facscanto instrument
1
Sox2 Expression Analysis by Flow Cytometry
2
Comprehensive Lymphocyte Immunophenotyping
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Lymph nodes and spleen were disintegrated into single cells as previously described45 (link) and stained for surface markers with the following antibodies: TCR-β (H57-597), B220 (R43-6B2), CD4 (RM4-5), and CD8 (53-6.7), and all were purchased from BD Biosciences (Franklin Lakes, NJ, USA) or eBioscience (San Diego, CA, USA).
Spleen cells were stimulated as previously described46 , and then stained for intracellular cytokines using a Mouse FOXP3 Buffer Set (BD Biosciences). The following fluorochrome-conjugated antibodies were used: CD4 (GK1.5), CD25 (7D4), CD3 (145-2C11), FOXP3 (FJK-16s), RORγt (AFKJ-9), IFNγ (XMG1.2), and IL-17 (eBio17B7), and all were purchased from BD Biosciences or eBioscience.
Data was collected with a FACSCanto instrument (BD Biosciences).
Spleen cells were stimulated as previously described46 , and then stained for intracellular cytokines using a Mouse FOXP3 Buffer Set (BD Biosciences). The following fluorochrome-conjugated antibodies were used: CD4 (GK1.5), CD25 (7D4), CD3 (145-2C11), FOXP3 (FJK-16s), RORγt (AFKJ-9), IFNγ (XMG1.2), and IL-17 (eBio17B7), and all were purchased from BD Biosciences or eBioscience.
Data was collected with a FACSCanto instrument (BD Biosciences).
3
Nasal Tissue Cell Isolation and Analysis
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Female BALB/cCrSlc mice (seven weeks of age) were administrated with PBS (vehicle) or liposomes (400 nmol/mouse) intranasally. Six hours after administration, single cells from nasal tissues were isolated by mechanical dissociation using stainless-steel mesh, followed by passing through 70 µm nylon mesh. The isolated cells were then treated with Red Blood Cell (RBC) lysing buffer (BioLegend, San Diego, CA, USA) to lyse red blood cells. After washing the cells with PBS, dead cells in the preparation were stained with 7-amino-actinomycin D (7-AAD) viability staining solution (BioLegend), according to the manufacturer protocol, and then analyzed using a FACSCanto instrument (BD Biosciences, San Jose, CA, USA).
4
Memory T Cell-Epithelial Interaction Assay
5
Immunophenotyping of Peripheral Blood Cells
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To determine percentages and numbers of T cells (CD3+CD19-), B cells (CD3-CD19+), CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), and natural killer (NK) cells (CD3-CD16+CD56+), peripheral blood samples (2 mL) from each subject were collected. For immunofluorescence staining, 50 µL of each blood samples were placed in TruCount tubes A nd B. Then, 20 µL of CD3-fluorescein isothiocyanate (FITC)/CD8-phycoerythrin (PE)/CD45-peridinin-chlorophyll protein (PerCP)/CD4-allophycocyanin (APC) antibodies (clones SK7/SK1/2D1/SK3, respectively) were added to tube A and 20 µL of CD3-FITC/CD16 + 56-PE/CD45-PerCP/CD19-APC antibodies (clones SK7/B73.1 NCAM16.2/2D1/SJ25C1, respectively) were added to tube B. All antibodies were purchased from BD Biosciences (San Jose, CA, USA). After incubation at room temperature for 20 minutes in the dark, stained cells were washed with 1× FACS buffer and then incubated for 15 minutes in the dark. Data on 15,000 cells were acquired on a FACSCanto instrument (BD Bioscience) and analyzed using MultiSET software.
6
Vi Antigen Expression on Salmonella
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Salmonella were grown to an OD600 of 0.5 in LB broth. Bacteria were washed twice and resuspended in PBS. Mouse anti-Vi antiserum from the Sclavo Behring Vaccines Institute of Global Health (SBVGH, formerly Novartis Vaccines Institute of Global Health) was added to the Salmonella at a final concentration of 1:800. Samples were incubated on ice for 1 hour followed by washing with PBS. Allophycocyanin-conjugated rabbit anti-mouse IgG (Sigma) was added at a final concentration of 1:800 and samples incubated for 1 h, washed, and resuspended in PBS/1% formaldehyde. Samples were analyzed by flow cytometry using a FACSCanto instrument (BD Biosciences) and FlowJo software (Tree Star, Inc). Salmonella were identified based on forward and side scatter characteristics and the level of Vi expression determined as the GMFI in the FL4 channel.
7
Multi-Parametric Flow Cytometry Analysis
8
Apoptosis Detection by Annexin V
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Cell death was assessed by phosphatidylserine externalization. Cell lines were stained with fluorescein-conjugated annexin V (BD Biosciences) and propidium iodide and analyzed on a BD FACSCalibur flow cytometer. Raw data were analyzed using the CellQuest Version 5.2.1 software. Results were normalized to survival of untreated cells. Flow cytometric immunophenotyping using fluorescently labeled monoclonal antibodies against ZAP70 and CD38 was performed at the Cleveland Clinic as part of diagnostic evaluation on a FACSCanto instrument (BD Biosciences). Staining protocols were standard lyses/washing protocols, as previously described [11 (link)].
9
Monocyte MR Expression in Alcoholic Hepatitis
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To investigate whether peripheral monocytes express MR and therefore could be a source of sMR, we studied the MR expression using flow cytometry. 20 patients with AH, 10 patients with AC, and 10 HCs was studied. Peripheral blood mononuclear cells were isolated by Ficoll-Hypaque (GE Healthcare Bio-sciences, Uppsala, Sweden) from EDTA whole blood samples. Samples were stored at -140°C until en bloc analyses. Cells were thawed in PBS containing 20% heat-inactivated pooled human AB-serum and blocked with 10 μl heat-inactivated mouse serum (Invitrogen, Carlsbad, California, USA) before staining with optimized amounts of fluorescent-conjugated antibodies; CD14-APC (clone MFP9; BD Bioscience, San Diego, CA), CD206-FITC (clone 19.2; BD Bioscience, San Diego, CA). Relevant isotype controls were included. The samples were analysed within 24 hours of preparation using a FACSCanto instrument (BD Bioscience). Data analyses were carried out using FlowJo v10.1 (Tree Star Inc., Ashland, OR). We report the relative fluorescent intensity (MFI) of sMR on the CD14+ monocytes.
10
Cell Cycle Profiling by Flow Cytometry
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Time course of cell-cycle profiles was performed by addition of 10% fetal bovine serum (FBS) medium 24 hours after serum deprivation. Neo- and HCaRG-Renca cells were harvested to proceed to nucleic acid staining. Fixed cells were incubated in phosphate buffered saline (PBS; Thermo Fisher Scientific) containing 0.5 mg/ml bovine pancreatic DNase-free RNase (11119915001; Sigma-Aldrich) and 50 µg/ml PI (P1304MP; Thermo Fisher Scientific) for 20 minutes at room temperature. Cells were analyzed using FACScanto instrument (BD Biosciences, Mississauga, Canada), and compiled with ModFit LT (Verity Software House, Topsham, ME, USA) and FACS DivaSoftware (BD Biosciences).
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