Approximately 1 × 106 cells/mL were harvested and washed with Hank’s balanced salt solution (HBSS) supplemented with 0.1% BSA. Cells were then resuspended with 0.1% BSA/HBSS containing PE-Cy5.5 coupled anti-Thy1.1 antibodies (clone HIS51, eBiosciences), or isotype control and incubated for 30 min at 4 °C. The cells were washed again by resuspending with 0.1% BSA/HBSS before being analyzed with an Accuri C6 benchtop flow cytometer (BD Biosciences, San Jose, CA, USA) as previously described [58 (link)]. Appropriate filter sets were used to detect GFP fluorescence or PE-Cy5.5 fluorescence.
Accuri c6 benchtop flow cytometer
Manufactured by BD
Sourced in United States
The Accuri C6 is a benchtop flow cytometer designed for cell analysis. It measures multiple parameters of individual cells or particles flowing through a laser beam, including size, granularity, and fluorescence. The Accuri C6 is capable of detecting and analyzing a wide range of cell types and is commonly used in various applications, such as immunology, cell biology, and drug discovery research.
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Lab products found in correlation
Hla c specific dt9 antibody, by Merck Group (2 mentions)
Anti cd107a texasred antibody, by Thermo Fisher Scientific (2 mentions)
Anti kir2dl2 3 apc gl183, by Beckman Coulter (2 mentions)
Anti kir3dl1 fitc dx9, by BioLegend (2 mentions)
Recombinant human il 2, by R&D Systems (2 mentions)
Lsrii cytometer, by BD (2 mentions)
Cytofix, by BD (2 mentions)
Cd25 apc clone pc61.5, by BioLegend (1 mentions)
Synthetic peptide, by GenScript (1 mentions)
7 protocols using accuri c6 benchtop flow cytometer
1
Thy1.1 Expression Profiling by Flow Cytometry
2
Peptide-mediated NK Cell Activation
3
Flow Cytometry Analysis of Immune Markers
4
Peptide Stabilization and NK Cell Cytotoxicity
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To test the effect of specific peptides, T2 cells were incubated in serum-free RPMI media overnight at 26°C in the presence of 100 μM synthetic peptide (GenScript). Cells were washed and stained with the HLA-C-specific DT9 antibody (EMD Millipore). Peptide stabilization was assessed by flow cytometry on an Accuri C6 benchtop flow cytometer (BD Biosciences).
NK cells were isolated from five healthy KIR A haplotype and HLA-C1 homozygous donors, and they were stimulated overnight with 500 units/mL recombinant human IL-2 (R&D Systems) in RPMI 10% complete media. T2 cells (3 × 104) were incubated with 100 μM peptide (Genscript) overnight in serum-free media at 26°C, washed, and re-suspended in serum-free media with anti-CD107a-TexasRed antibody (eBioscience). T2 cells were incubated with NK cells at a target:effector ratio of 10:1 for 3.5 hr in serum-free media with 500 units/mL recombinant human IL-2. Surface staining was performed using anti-KIR2DL2/3-APC GL183 (Beckman Coulter) and anti-KIR3DL1-FITC DX9 (BioLegend) to identify KIR2DL3+/KIR3DL1− NK cells. Following surface staining, cells were treated with live/dead orange (Life Sciences) and fixed (Cytofix, BD Biosciences). Cells were analyzed on an LSR II cytometer (BD Biosciences) at the Stanford Flow Cytometry Core Facility. A representative gating strategy is shown inFigure S5 B.
NK cells were isolated from five healthy KIR A haplotype and HLA-C1 homozygous donors, and they were stimulated overnight with 500 units/mL recombinant human IL-2 (R&D Systems) in RPMI 10% complete media. T2 cells (3 × 104) were incubated with 100 μM peptide (Genscript) overnight in serum-free media at 26°C, washed, and re-suspended in serum-free media with anti-CD107a-TexasRed antibody (eBioscience). T2 cells were incubated with NK cells at a target:effector ratio of 10:1 for 3.5 hr in serum-free media with 500 units/mL recombinant human IL-2. Surface staining was performed using anti-KIR2DL2/3-APC GL183 (Beckman Coulter) and anti-KIR3DL1-FITC DX9 (BioLegend) to identify KIR2DL3+/KIR3DL1− NK cells. Following surface staining, cells were treated with live/dead orange (Life Sciences) and fixed (Cytofix, BD Biosciences). Cells were analyzed on an LSR II cytometer (BD Biosciences) at the Stanford Flow Cytometry Core Facility. A representative gating strategy is shown in
5
Flow Cytometry Staining and Analysis
6
NF-κB Activation Dynamics in C57E Cells
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C57E cells isolated from WT C57bl/6 aorta endothelium and immortalized with a py-MT lentiviral plasmid were used. Cells contain an NF-κB binding sequence upstream of a GFP reporter gene in their genome. The cells containing this construct constitutively express red fluorescent protein and only express GFP when NF-κB is released from its receptor protein [inhibitor of NF-κB (IκB-α)]. Cells were seeded in a 24-well plate at a density of 40,000 cells per well with 500 μl of media per well. Twenty-four hours later, PIP3 was added at a dose of 0, 10, or 20 μM. The positive control received 100 ng of LPS. Eight and 12 hours later, cells were analyzed via flow cytometry using a BD Accuri C6 Benchtop Flow Cytometer (fig. S1, H to J).
7
Validation of Cell Viability via Flow Cytometry
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Flow cytometry was used to validate the viability results. Cells were cultured in T25 cell culture flasks following the aforementioned protocol. Control cells (N = 3) and cells stored in the optimal additive combination (1% sericin, 5 mmol/L adenosine, 50 μg/mL L-ascorbic acid and 1 mM allopurinol) (N = 3) for three days were compared. Propidium iodide (PI), which binds to double-stranded DNA of dead cells, was added to the culture medium of both culture groups at a concentration of 2.5 μg/300 μL sample and cells were returned to the incubator for 15 minutes. Cells were then rinsed with PBS, trypsinized for 2–3 minutes, then washed and re-suspended in ice-cold HBSS +4% FBS. Samples were kept on ice and analyzed using the BD Accuri C6 bench top flow cytometer. PI is excited by the 588 nm laser and is detected in filter 616//23 (FL3).
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