The following antibodies and dyes were used for FACS analysis of human cells: CD4 (RPA-T4), Gata-3 (16E10A23), T-bet (4B10) (all BioLegend), and dead cell marker Viability Dye eFluor™ 780 (eBioscience). The following antibodies and dyes were used for FACS analysis of mouse cells: CD4 (RM4-5), Thy1.1 (Ox-7), Thy1.2 (30-H12), Gata-3 (16E10A23), IL-4 (11B11), IFNγ (XGM1.2), T-bet (eBio4B10) (all BioLegend), and dead cell marker Viability Dye eFluor™ 780 (eBioscience). For intracellular cytokine analysis, cells were restimulated with 5 ng/ml PMA and 500 ng/ml ionomycin for 4 h. 10 µg/ml BrefA were added after 2 h. Stainings were performed with up to 106 cells from PBMC, lymph node, or erythrocyte depleted spleen cells, in 50 µl of FACS buffer (PBS/0.1% bovine serum albumin/0.02% NaN3). After surface staining (30 min, 4°C), cells were fixed for 30 min at 4°C (fixation buffer, eBioscience), permeabilized (permeabilization buffer, eBioscience), and intracellularly stained for Gata-3 and T-bet or IL-4 and IFNγ expression for 45 min at room temperature. The cells were analyzed on a BD™ LSR II flow cytometer with the use of FACS Diva software (all Becton Dickinson). For further analyses of the data, FlowJo (TreeStar Inc.) software was used. Median fluorescence intensity (MFI) ratios of T-bet and Gata-3 were calculated by dividing the median fluorescence intensities of the two markers.
Viability dye efluor 780
Manufactured by Thermo Fisher Scientific
Sourced in United States
Viability Dye eFluor™ 780 is a fluorescent dye used for the identification of viable cells in flow cytometry applications. It exhibits excitation at 780 nm and emission at 820 nm. The dye is designed to stain dead cells, allowing them to be excluded from the analysis.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (7 mentions)
Facsdiva software, by BD (4 mentions)
Lsrfortessa flow cytometer, by BD (3 mentions)
Lsrfortessa, by BD (3 mentions)
Lsr 2 flow cytometer, by BD (3 mentions)
Ic fixation buffer, by Thermo Fisher Scientific (3 mentions)
Facscanto, by BD (2 mentions)
Cell stimulation cocktail, by Thermo Fisher Scientific (2 mentions)
Fixation buffer, by BioLegend (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (2 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (2 mentions)
Facslyric, by BD (2 mentions)
Anti γδ tcr pe, by BD (2 mentions)
Lsr fortessa cytometer, by BD (2 mentions)
Fixation permeabilization reagent, by Thermo Fisher Scientific (2 mentions)
Celltrace violet ctv, by Thermo Fisher Scientific (2 mentions)
Cd4 apc clone rpa t4, by BioLegend (2 mentions)
Anti ly6g clone 1a8, by BD (1 mentions)
Anti cd11b clone m1 70, by Thermo Fisher Scientific (1 mentions)
56 protocols using viability dye efluor 780
1
FACS Analysis of T Cell Subsets
2
Murine Immune Cell Profiling
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Anti-mouse CD16/32 (clone 2.4G2, BD Pharmingen, BD, New Jersey, U.S.), anti-Ly6G (clone 1A8, BD Pharmingen, BD, New Jersey, U.S.), anti-CD11b (clone M1/70, eBioscience, Affymetrix, California, U.S.). Viability Dye eFluor™ 780 (eBioscience, Affymetrix, California, U.S.) or DAPI (BioLegend, California, U.S.) were used to determine viable cells.
3
Multicolor Flow Cytometry Analysis of Immune Cell Populations
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Anti-GD2 (clone 14.g2a) mAb was purified and filter sterilized in PBS. Total mouse IgG control Ab was obtained from Jackson Immunoresearch. Directly labeled mAbs used for staining were CD11b-A700 (M1/70), H-2Kb/H-2Db-PE (28-8-2006), F4/80-PE-Cy7 (BM8), CD11c-PerCP (N418), CD4-PerCP (L3T4), Ly-6G-PE-Cy7 (1A8), CD64-PE (X54-5/7.1), CD16/CD32-A647 (2.4G2), CD206-APC (MR5D3) from Biolegend, CD45.2-FITC (104), CD11c-APC (HL3), NK1.1-PE (PK136), Ly-6C-PE (AL-21) and CD3-APC (145-2C11) from BD, MHC II-PE (M5/114.15.2) and FoxP3-PE-Cy7 (FJK-16s) from eBioscience, CD8-A700 (53–6.7) from Exbio. Cells for staining were washed in PBS, incubated with Viability Dye eFluor 780 (eBioscience), resuspended in PBA, transferred to a V-bottom 96-wells plate and stained using specific mAb or the appropriate isotypes. Cells analyzed on a Cyan apparatus (Beckman Coulter) using Summit software.
4
Suppression of Teff Cell Proliferation
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Human Teff cells (CD4+CD25lowCD127high) were sorted from healthy donor PBMCs using BD FACS AriaII and were labeled with 5 μM CellTrace Violet (CTV, Invitrogen C34557) for 20 min at 37°C. Human in vitro expanded Treg cells or iTreg cells were diluted at various concentrations and were incubated with Teff cells that were labeled with CTV and activated with Dynabeads Human T-activator CD3/CD28 (Teff cells: beads = 4:1) in a U-bottom 96-well-plate. Ninety hours later, cells were harvested, incubated with Viability Dye eFluor 780 (eBioscience 65-0865-14), and examined for Teff cell proliferation on BD LSRFortessa X-20.
5
Analysis of IRF4 Expression in Human CD4+ T Cells
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Human CD4+ T cells were incubated with PBS in the presence of a viability dye (Viability Dye eFluor 780 eBioscience 65-0865-18) before being washed with flow cytometry staining buffer (eBioscience 00-4222-26), then fixed and permeabilized using a Transcription Factor Staining Buffer Set (eBioscience 00-5523-00). Cells were then incubated with Anti-Human/Mouse IRF4 PE-Cyanine7 (eBioscience 25-9858-82) and washed. Cells were analyzed on a Millipore Guava Flow Cytometer 8HT and the machine was compensated using appropriate single color controls. Analysis of flow cytometry data files was performed using FlowJo version 10.0.7.
6
Flow Cytometric Analysis of Cell Differentiation
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For the flow cytometric analysis of cell differentiation, cells were stained with anti-CD14 (BV785, clone M5E2, BioLegend, San Diego, CA, USA), anti-CD33 (Thermo Fisher Scientific, Waltham, MA, USA), anti-CD34 (Thermo Fisher Scientific, Waltham, MA, USA), and viability dye eFluor780 (eBioscience, San Diego, CA, USA).
Cells were stained with viability dye eFluor506 (eBioscience, San Diego, CA, USA), and intracellular NOTCH1 expression was detected with anti-NOTCH1 (PE, clone mN1A, BioLegend, San Diego, CA, USA) using the eBioscience Intracellular Fixation & Permeabilization Buffer Set (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. All measurements were performed with a BD LSRFortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with FlowJo Version 10.8 (BD Biosciences, Franklin Lakes, NJ, USA).
Cells were stained with viability dye eFluor506 (eBioscience, San Diego, CA, USA), and intracellular NOTCH1 expression was detected with anti-NOTCH1 (PE, clone mN1A, BioLegend, San Diego, CA, USA) using the eBioscience Intracellular Fixation & Permeabilization Buffer Set (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. All measurements were performed with a BD LSRFortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with FlowJo Version 10.8 (BD Biosciences, Franklin Lakes, NJ, USA).
7
Flow Cytometric Analysis of CD4+ and CD8+ T Cells
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Frozen PBMCs were thawed and washed with RPMI 1640 supplemented with 10% foetal calf serum (FCS), 2 mM L-Glutamine, 10 mM HEPES, and 50 U/ml Penicillin-Streptomycin (all from Thermo Fisher). Cells were stained with Viability Dye eFluor 780 (eBioscience) and antibodies against CD3 (PE-labelled, clone HIT3a, BD Biosciences), CD4 (BrilliantViolet510-labelled, clone OKT4, BioLegend), CD8 (PerCP-Cy5.5-labelled, clone HIT8a, BioLegend), CD25 (PE-Cy7-labelled, clone 2A3, BD Biosciences) and CD127 (AlexaFluor647-labelled, clone HIL-7R-M21, BD Biosciences). After cell wash, PBMCs were fixed with Fixation Buffer (BioLegend) and subsequently analysed using a BD LSRFortessa flow cytometer (BD Biosciences). Gating procedures are depicted in S3 Fig . For detection of mIL-7R in the second independent cohort of tuberculosis patients and healthy contacts we used the CD127 antibody clone A019D5 (BioLegend). Comparison of both antibody clones revealed similar T-cell binding pattern as well as percentages of mIL-7Rhigh and mIL-7Rlow T cells.
8
Flow Cytometric Analysis of Regulatory T Cells
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The following anti-human antibodies were used: CD4-PE/Cy5, CD25-FITC, and Foxp3-Alexa647 from BioLegend (San Diego, CA, USA). Mouse cells were stained with fluorochrome-labeled mAbs to CD4-Pacific Blue, Brilliant Violet 605 and Alexa-700 (RM4-5), CD8-Pacific Blue (53-6.7), CD25-APC (7D4), Ki-67-PE (Ki-67), and Thy1.1-PercP (Ox-7) from BioLegend. Foxp3-APC (FJK-16s), dead cell marker Viability Dye eFluor™ 780, and staining reagents from eBioscience (San Diego, CA, USA) were used according to the manufacturer’s instructions. To analyze expression of surface proteins, the cells were stained with the appropriate antibodies for 20 min at 4°C, washed once with FACS buffer (PBS, 0.1% BSA, and 0.02% NaN3), and fixed with 2% paraformaldehyde. For intracellular staining of Foxp3 and Ki-67, the cells were first surface stained, permeabilized with Fix/Perm (eBioscience), and stained with Foxp3 and Ki-67 antibodies diluted in Perm/Wash (eBioscience). To calculate absolute Treg numbers, unlabeled microbeads (BD Biosciences, Franklin Lakes, NJ, USA) were added to the stained cells and the following formula was used: absolute Treg numbers = (beads used × Treg events)/beads measured. Acquisition was performed on a BD™ LSR II or FACSCalibur, and data were analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA).
9
Quantifying Apoptosis and Tumor Immunity
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For apoptosis assay, CRC cells were harvested, and apoptosis was assessed by flow cytometry using an Annexin V Apoptosis Detection Kit (eBioscience) at 48 h post-transfection. For TME immune microenvironment analysis, subcutaneous tumors were cut into 2-mm3 pieces. The tissues were digested and incubated with collagenase D for 30 min. All cells were washed in PBS with 2% FBS. Then the cells were stimulated with PMA (50 ng/mL), ionomycin (1 mM), Golgi Stop, and Golgi Plug for 4 h before cytokine detection. Next, cells were incubated with Viability Dye eFluor 780 (eBioscience, 1:1,000, #65-0865-14) and antibodies TCR β chain (Biolengend, 1:200, #109221), CD8 (BD, 1:200, #563898), CD45 (eBioscience, 1:200, #11-0451-85), IFN-γ (eBioscience, 1:200, #12-7311-82). All samples were run on the BD LSRFortessa Flow Cytometer (BD Biosciences), and FlowJo software (TreeStar, Ashland, OR, USA) was used to analyze the data.
10
Isolation of Brain and Spinal Cord Cells from LysM tdRFP Mice
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To isolate cells from the whole brain and spinal cord of LysM tdRFP mice, the tissue was homogenized after PBS perfusion, and a percoll gradient was performed according to standard protocols with 25 and 75% stock istotonic percoll (GE Helthcare) and HBSS. Cells were blocked with antibodies to Fcγ receptors (DRFZ, clone 2.4G2) to avoid non-specific staining, and were subsequently stained with FITC-labeled PerCP-labled rat anti-CD45 (BioLegend) or Cy5- (DRFZ), APC- or Pacific Blue™ (BioLegend)-labeled rat anti-CD11b, in some experiments fixable Viability Dye eFluor®780 (eBioscience), anti-CX3CR1 APC and anti-CD3 Brilliant Violet™ (both BioLegend) were used according to standard procedures, followed by fixation using 4% Paraformaldehyde (Electron Microscopy Science) for 10 min. FACS analysis was performed on a LSR Fortessa (BD).
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