The differentiated Th2 cells were plated in 96-well plates at density of 1 × 106 cells/mL and treated with GJExCR and Cell Activation Cocktail (R&D systems Inc.; Cat. No. #5476) for 24 h. After incubation, the culture medium of Th2 cells was harvested and cytokine secretion was analyzed using a LEGENDplex™ Mouse Th Cytokine Panel (12-plex) assay kit (BioLegend, San Diego, CA, USA) and LSRFortessa X-20 flow cytometry (BD Biosciences).
Lsrfortessa x 20 flow cytometry
Manufactured by BD
Sourced in United States
The BD LSRFortessa™ X-20 is a flow cytometry system designed for high-performance multi-parameter analysis. It features a 20-parameter configuration, enabling researchers to conduct comprehensive cellular analysis.
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Lab products found in correlation
Percp cy5.5 conjugated anti cd4, by BioLegend (3 mentions)
Apc cy7 conjugated anti cd3, by BD (3 mentions)
Live dead fixable aqua, by Thermo Fisher Scientific (3 mentions)
Brilliant violet 421 conjugated anti cd25, by BD (2 mentions)
Brilliant violet 605 conjugated anti cd8, by BioLegend (2 mentions)
Fitc conjugated anti pd 1, by BioLegend (2 mentions)
Apc conjugated anti cd127, by BioLegend (2 mentions)
Pe conjugated foxp3 antibody, by Thermo Fisher Scientific (2 mentions)
Pe cy7 conjugated anti cd45ra, by BD (2 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions)
Cell activation cocktail, by R&D Systems (1 mentions)
Anti gata3 bv421, by BD (1 mentions)
Anti cd3 fitc, by BD (1 mentions)
Anti cd4 fitc, by BD (1 mentions)
Anti cd4 bb700, by BD (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Live dead fixable viability stain 510, by BD (1 mentions)
Anti cd45 bv786 clone 30f 11, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
8 protocols using lsrfortessa x 20 flow cytometry
1
Analyzing Th2 Cytokine Secretion
2
Immune Cell Profiling in Allergic Dermatitis
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Immune cells were isolated from the spleen and lymph node of OVA-induced AD mice. The cells were stained with anti-CD4-FITC and anti-CD8-APC antibodies (BD Biosciences, San Jose, CA, USA). The differentiated Th2 cells were stained with anti-CD3-FITC, anti-CD4-BB700, and anti-GATA3-BV421 (BD Biosciences). After staining, the populations of CD4+ T cells, CD8+ T cells, and Th2 cells were analyzed using a LSRFortessa™ X-20 flow cytometry (BD Biosciences) and FlowJo v. 10 software (FlowJo, Ashland, OR, USA).
3
Analyzing Host Immune Responses to Salmonella Infection
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Six- to eight-week-old female C57BL/6 WT mice were anesthetized and challenged i.g. with either 1 × 105 CFU of S. Typhimurium or an equal volume of PBS (as uninfected control). At day 4 post-infection, mice were euthanized and spleens, livers, and small intestine were collected and single-cell suspensions were prepared for flow cytometry, as described above. Cells suspensions were stimulated with 10 µg/ml brefeldin A (Sigma-Aldrich) for 5 h prior to intracellular staining. Then, cells were stained with anti-CD45-BV786 (clone 30-F11; BD PharMingen), anti-TCR-β-PE-Cy7 (clone H57-597; BD PharMingen), anti-CD4-APC-H7 (clone RM4-5; BD PharMingen), anti-CD25-BV421 (clone PC61; BD PharMingen). After staining, cells were fixed and permeabilized (Cytofix/Cytoperm kit; BD PharMingen) and, subsequently, stained intracellularly with anti-IL10 (clone JES5-16E3, BD PharMingen) and anti-Foxp3 (clone PE-CF594, BD PharMingen). All FACS samples were first stained with LIVE/DEAD fixable Viability Stain 510 (BD PharMingen). The intracellular stain was controlled by FMO and the analysis was performed using a LSR Fortessa X-20 Flow cytometry (BD Biosciences).
4
Measurement of AuNPs Uptake in A549 Cells
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For the measurement of AuNPs uptake using flow cytometry, A549 cells were seeded in six well plates at a density of 2 × 105 cells/well in 3 mL of CCM and allowed to adhere overnight at 37 °C in 5% CO2. Then, the cells were exposed to different sizes of AuNPs at concentrations of 5, 15, and 30 µg/mL while the control cells were treated with CCM for 24 h. After the exposure time, the cells were washed with PBS three times to remove any unattached AuNPs and were then harvested and centrifuged for 5 min at 1500 rpm at 4 °C. The supernatant was discarded, and the cell pellet was resuspended in 500 µL of PBS. The samples were then analysed using BD LSRFortessa™ X-20 flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA)) by acquiring at least 10,000 events.
5
Quantifying Oxidative Stress in A549 Cells
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The CellRox™ deep red reagent kit was used for oxidative stress detection in A549 cells as per the manufacturer’s instructions. A549 cells were seeded in 12 well plates at a density of 2 × 104 cells/well and incubated overnight to adhere. The cells were then treated with 5, 15, and 30 µg/mL of different sizes of AuNPs for 24 h while a 50 µM menadione treatment for 1 h was used as the positive control. Following the incubation, the cells were detached by trypsinisation and centrifuged at 1500 rpm for 5 min. The cells were then stained with 5 µM CellRox deep red reagent diluted in phenol-free RPMI 1640 media for 1 h at 37 °C and protected from light. The cells were then centrifuged for 5 min at 1500 rpm after the staining and the pellet was resuspended with 1 mL of RPMI 1640 media. The amount of ROS generated was then analysed immediately by BD LSRFortessa™ X-20 flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) by acquiring at least 10,000 events.
6
Multi-color Flow Cytometry of PBMCs
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Multi-color flow cytometry of PBMCs was performed at CNUH. The following human antibodies were used for multi-color flow cytometry: Brilliant Violet 421-conjugated anti-CD25, PE-Cy7 conjugated anti-CD45RA, and APC-Cy7 conjugated anti-CD3 (all from BD Biosciences, San Jose, CA, USA) and Brilliant Violet 605-conjugated anti-CD8, PerCP-Cy5.5-conjugated anti-CD4, FITC-conjugated anti-PD-1, and APC-conjugated anti-CD127 (all from BioLegend, San Diego, CA, USA). For intracellular staining of FOXP3, cells were fixed and permeabilized after surface staining using the Foxp3/Transcription factor staining buffer set (eBioscience) and incubated with PE-conjugated FOXP3 antibody (eBioscience, San Diego, CA, USA). To exclude dead cells, single-cell suspensions were first incubated for 20 min in viability dye (LIVE/DEAD Fixable Aqua, Thermo Fisher). Stained cells were analyzed using BD LSR Fortessa X-20 flow cytometry (BD Biosciences). Fluorescence-activated cell sorting (FACS) analysis was performed using FlowJo software (Tree Star, Ashland, OR, USA).
7
Multi-color Flow Cytometry for PBMC Profiling
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Multi-color flow cytometry of PBMCs was performed at CNUH. The following human antibodies were used for multi-color flow cytometry: Brilliant Violet 421-conjugated anti-CD25, PE-Cy7 conjugated anti-CD45RA, and APC-Cy7 conjugated anti-CD3 (BD Biosciences, San Jose, CA, USA), and Brilliant Violet 605-conjugated anti-CD8, PerCP-Cy5.5-conjugated anti-CD4, FITC-conjugated anti-PD-1, and APC-conjugated anti-CD127 (BioLegend, San Diego, CA, USA). For intracellular staining of FOXP3, cells were fixed and permeabilized after surface staining using the Foxp3/Transcription factor staining buffer set (eBioscience) and incubated with a PE-conjugated FOXP3 antibody (eBioscience, San Diego, CA, USA). To exclude dead cells, single-cell suspensions were first incubated for 20 min in a viability dye (LIVE/DEAD Fixable Aqua, Thermo Fisher). The stained cells were analyzed using BD LSR Fortessa X-20 flow cytometry (BD Biosciences). Fluorescence-activated cell sorting was performed using FlowJo software (Tree Star, Ashland, OR, USA).
8
Multicolor Flow Cytometry of PBMCs
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Multicolor flow cytometry of PBMCs was performed at CNUH. The following human antibodies were used for multi‐color flow cytometry: APC‐Cy7‐conjugated anti‐CD3, PE‐CF594‐conjugated anti‐PD1 (both from BD Pharmingen), PE‐conjugated anti‐CXCR3 (CD183), PerCP‐Cy5.5‐conjugated anti‐CD4, APC‐conjugated anti‐CCR6 (CD196), BV421‐conjugated anti‐CCR4 (CD194), and BV605‐conjugated anti‐CD8 (all from BioLegend). To exclude dead cells, single‐cell suspensions were first incubated for 20 min in viability dye (LIVE/DEAD Fixable Aqua, Thermo Fisher Scientific). Stained cells were analyzed using BD LSR Fortessa X‐20 flow cytometry (BD Biosciences). Fluorescence‐activated cell sorting (FACS) analysis was performed using FlowJo software (Tree Star).
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