After stimulation, NK cells were co-cultured with YAC-1 target cells (mouse lymphoma lymphoblast fibroblast; ATCC TIB-160; authenticated and mycoplasma tested) at the indicated effector cell:target cell (E:T) ratios for 4 h (37 °C and 5% CO2). Co-cultures were then stained with Fixable Viability Dye™ (FVD) eFluor™ 506 (eBioscience) and fluorochrome-conjugated antibodies specific for cell surface markers (CD45, CD3 and NK1.1) to differentiate NK and YAC-1 cells and analyzed by flow cytometry. NK cell only and YAC-1 only controls were used to determine spontaneous lysis of target cells and set gating strategy. Percent specific lysis was calculated as the proportion of YAC-1 cells (FSCint SSCint-hi CD45+ CD3+ CD49b-) positive for FVD, minus spontaneous lysis (Littwitz-Salomon et al, 2018 (link); Valiathan et al, 2012 (link)).
Fixable viability dye fvd efluor506
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fixable viability dye (FVD)-eFluor506 is a fluorescent dye used to identify and quantify dead cells in flow cytometry experiments. It binds to proteins on the surface of dead cells, allowing for their discrimination from live cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd14 fitc, by BioLegend (2 mentions)
Anti nkp46 pe cy7, by BioLegend (2 mentions)
Anti cd19 apc cy7, by BioLegend (2 mentions)
Anti cd3 percp cy5, by Thermo Fisher Scientific (2 mentions)
Anti ulbp4 mab clone 709116, by R&D Systems (2 mentions)
Streptavidin bv421 sa bv421, by BioLegend (2 mentions)
Goat anti mouse igg pe gam pe, by BioLegend (2 mentions)
Anti cd3 pacific blue clone ucht1, by BD (1 mentions)
Anti cd8 pe cy7 clone sk1, by BioLegend (1 mentions)
Anti il 2 pe clone mq1 17h12, by BioLegend (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Brefeldin a, by BD (1 mentions)
Anti γ h2ax ab11174, by Abcam (1 mentions)
5 protocols using fixable viability dye fvd efluor506
1
NK Cell-Mediated Cytotoxicity Assay
2
Intracellular Cytokine Profiling of SARS-CoV-2 Specific T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate intracellular cytokine production, PBMC were stimulated with PepMix peptide pools of spike glycoprotein, NCAP and VME1 (1 μg of each peptide pool/mL) overnight at 37°C, 5% CO2 in the presence of brefeldin A at 10 μg/mL (BD Biosciences). PBMC cultured with medium alone or PMA/IONO were used as negative and positive controls, respectively. After the stimulation, PBMC were washed and stained for 20 min at 4°C in PBS with antibodies following an additional staining with Fixable viability Dye (FvD)-eFluor 506 (eBioscience) for 10 min at 4°C. T cell phenotype was investigated performing surface staining with anti-CD3-Pacific Blue (clone UCHT1; BD), anti-CD4-Alexa Fluor 488 (clone RPA-T4; Biolegend), anti-CD8-PE-Cy7 (clone SK1; Biolegend), antibodies. Then, PBMC were permeabilized and fixed with the Cytofix/Cytoperm kit according to the manufacturer's instructions (BD Biosciences). Intracellular staining was performed using anti-IFN-γ APC (clone 4S.B3; Biolegend), anti- TNF-α APC AF700 (clone IPM2, Beckman Coulter), anti-IL-2 PE (clone MQ1-17H12, Biolegend) antibodies for 30 min at 4°C. Samples were directly acquired on a Cytoflex (Beckman Coulter) and analyzed with Kaluza software.
3
Antibody Panel for ULBP4 Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-ULBP4 mAb (clone 709116) was purchased from R&D (Minneapolis, MN, USA). The ULBP4-specific mAb DUMO1 was previously described [32 (link)]. Anti-CD3-PerCP-Cy5.5 and fixable viability dye(FVD)-eFluor506 were from eBioscience (SanDiego, CA, USA), anti-CD19-APC-Cy7, anti-CD14-FITC, anti-NKp46-PE-Cy7, streptavidin-BV421 (SA-BV421) and goat-anti-mouse-IgG-PE (GaM-PE) were from Biolegend (SanDiego, CA, USA).
4
Immunoblotting and Flow Cytometry Analyses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies to AIM2 (13095s), p-IRF3-S396 (4947s), IRF3 (4302s), p-TBK1 (5483s), TBK1 (3013s), p-AKT1 (9018s), AKT1 (2938s), p-AKT2 (8599s), AKT2 (3063s), p-AKT (4060P), and AKT3 (14982s) were purchased from Cell Signaling Technology. Antibodies to p-IRF3-S385 (D151514) were purchased from Sangon Biotech. Anti–ionized calcium binding adapter molecule 1 (Iba1; 019–19741) was from Wako. Anti-Apoe (ab1906) and anti-γH2AX (ab11174) were from Abcam. Anti-CD11b (101204) was from Biolegend. Anti-AKT (21054) was from SAB. Antibodies to actin (A1978) and DNA-PK (SAB4502385) were purchased from Sigma-Aldrich. Anti–CD45-FITC (30-F11,11-0451-82), anti–CD45-Alexa Fluor 700 (30-F11,56–0451-82), anti–CD8a-PE (53–6.7,12-0081-83), anti–CD11b-APC (M1/70,17-0112-82), anti–IL17-PE (eBio18B7,12-7177-81), anti–IFNγ-PerCP-Cyanine5.5 (XMG1.2,85-45-7311-82), anti–IFNγ-APC (XMG1.2,17-7311-82), and Fixable Viability Dye (FVD) eFluor 506 were from eBioscience. Anti–CD4-APC-Cy7 (GK1.5,100414) was from Biolegend. Pertussis toxin (#180) was from List Biological Laboratories. Mycobacterium tuberculosis H37Ra (231141) was from BD. Incomplete Freund’s adjuvant (F5506) was from Sigma-Aldrich. MOG35-55 peptide (residues 35–55, Met-Glu-Val-Gly-Trp-Tyr-Arg-Ser-Pro-Phe-Ser-Arg-Val-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys) was synthesized by Sangon Biotech (Shanghai).
5
Flow Cytometry Analysis of ULBP4
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-ULBP4 mAb (clone 709116) was purchased from R&D (Minneapolis, MN, USA). The ULBP4-specific mAb DUMO1 was previously described (Zoller et al., 2018) (link). Anti-CD3-PerCP-Cy5.5 and fixable viability dye(FVD)-eFluor506 were from eBioscience (SanDiego, CA, USA), anti-CD19-APC-Cy7, anti-CD14-FITC, anti-NKp46-PE-Cy7, streptavidin-BV421 (SA-BV421) and goat-anti-mouse-IgG-PE (GaM-PE) were from Biolegend (SanDiego, CA, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!