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Live dead fixable aqua staining kit

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The Live/Dead Fixable Aqua Staining Kit is a fluorescent staining solution used to identify and differentiate live and dead cells in a sample. It provides a rapid and sensitive method for assessing cell viability.

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Lab products found in correlation

Golgi stop and golgi plug, by BD (2 mentions) Fitc conjugated anti cd107a, by BioLegend (2 mentions) Pecy7 conjugated anti cd56, by BioLegend (2 mentions) Bv650 conjugated ifnγ 93705 4s b3, by BioLegend (2 mentions) Bv421 conjugated tnfα 562783 mab11, by BD (2 mentions) Cd4 alexa fluor 700, by Thermo Fisher Scientific (2 mentions) Ifn γ efluor450, by Thermo Fisher Scientific (2 mentions) Il2 percpefluor710, by Thermo Fisher Scientific (2 mentions) Foxp3 alexa fluor 488, by BioLegend (2 mentions) Cd127 pe, by Miltenyi Biotec (2 mentions) Foxp3 buffer set, by Thermo Fisher Scientific (2 mentions) Streptavidin alexa fluor 700, by Thermo Fisher Scientific (2 mentions) Brilliant violet 421 cd25, by BioLegend (2 mentions) Streptavidin ecd, by Beckman Coulter (2 mentions) Cd45ro ecd, by Beckman Coulter (2 mentions) Countbright absolute counting beads, by Thermo Fisher Scientific (2 mentions) Pd 1 clone j105, by Thermo Fisher Scientific (2 mentions) Cd3 clone okt3, by Thermo Fisher Scientific (2 mentions) Lsr 2 fortessa cytometer, by BD (2 mentions) Intracellular staining buffer, by BioLegend (1 mentions)

7 protocols using live dead fixable aqua staining kit

1

Assessing Natural Killer Cell Function

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Assessment of NK cell function by flow cytometry was carried out as previously described(30 ). Briefly, PBMCs (n=6) and targets cells were co-cultured and stained with FITC conjugated anti-CD107a (93937, BioLegend). An hour later, Golgi Stop and Golgi Plug (BD Biosciences) was added and cells were incubated for 3 hours. Cells were stained with Live/Dead Fixable Aqua Staining Kit (Thermo Fisher), PE-CY7 conjugated anti-CD56 and PE-CF594 conjugated anti-CD3, PE conjugated anti-CD69 (310906 (FN50), BioLegend), fixed and permeabilized. Permeabilized cells were stained with BV650 conjugated IFNγ (93705 (4S.B3), BioLegend) and BV421 conjugated TNFα (562783 (Mab11), BD Biosciences).
Arvindam U.S., van Hauten P.M., Schirm D., Schaap N., Hobo W., Blazar B.R., Vallera D.A., Dolstra H., Felices M, & Miller J.S. (2020). A trispecific killer engager molecule against CLEC12A effectively induces NK cell mediated killing of AML cells. Leukemia, 35(6), 1586-1596.
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2

Assessing Natural Killer Cell Function

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Assessment of NK cell function by flow cytometry was carried out as previously described(30 ). Briefly, PBMCs (n=6) and targets cells were co-cultured and stained with FITC conjugated anti-CD107a (93937, BioLegend). An hour later, Golgi Stop and Golgi Plug (BD Biosciences) was added and cells were incubated for 3 hours. Cells were stained with Live/Dead Fixable Aqua Staining Kit (Thermo Fisher), PE-CY7 conjugated anti-CD56 and PE-CF594 conjugated anti-CD3, PE conjugated anti-CD69 (310906 (FN50), BioLegend), fixed and permeabilized. Permeabilized cells were stained with BV650 conjugated IFNγ (93705 (4S.B3), BioLegend) and BV421 conjugated TNFα (562783 (Mab11), BD Biosciences).
Arvindam U.S., van Hauten P.M., Schirm D., Schaap N., Hobo W., Blazar B.R., Vallera D.A., Dolstra H., Felices M, & Miller J.S. (2020). A trispecific killer engager molecule against CLEC12A effectively induces NK cell mediated killing of AML cells. Leukemia, 35(6), 1586-1596.
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3

NK Cell Degranulation and Cytokine Assay

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PBMCs were plated overnight and then co-cultured for 4 h with HL-60 at an effector/target ratio of 2:1 with the noted treatments. FITC-conjugated anti-CD107a (clone H4A3; BioLegend), used to evaluate NK cell degranulation, was added at start of the 4-hour incubation period. After 1-hour incubation, Golgi Stop and Golgi Plug (BD Biosciences) were added for the last 3 h. At the end of the 4 h, the cells were stained using the Live/Dead Fixable Aqua Staining Kit (Thermo Fisher Scientific), surface stained with PE-Cy7-conjugated anti-56 (clone HCD56; BioLegend) and PE-CF594-conjugated anti-3D (clone 3G8; BioLegend), fixed with 2% paraformaldehyde, and permeabilized with intracellular staining buffer (BioLegend). The cells were stained for BV650-conjugated IFNγ (clone 4S.B3; BioLegend). NK cells were identified as CD56+/CD3-/live cells and gated into CD56 bright and CD56 dim fractions. Cells were run on LSRII (BD Biosciences) for 60 s per sample, analyzed with FlowJo software, and graphed on GraphPad Prism software.
Baik J., Felices M., Yingst A., Theuer C.P., Verneris M.R., Miller J.S, & Perlingeiro R. (2020). Therapeutic effect of TRC105 and decitabine combination in AML xenografts. Heliyon, 6(10), e05242.
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4

Comprehensive Immune Phenotyping Protocol

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All staining experiments were performed at 4°C for 30 minutes. Antibodies used were CD3-PerCPCy5.5, CD3-AF700, CD4-Brilliant Violet 605, CD8-APC-Cy7, CD25-APC, CD134-PE, OX40-APC, TNF-α-PECy7, CD154-APC, CD152-PCF594, Tbet-PerCPCy5.5, PD1-PeCy7, CD15s-Brilliant Violet 421, Helios-PE, ICOS-PeCy7, CD39-Brilliant Violet 711 (Becton Dickinson (BD) Biosciences), CD4-Alexa Fluor 700, IFN-γ-eFluor450, IL2-PerCPeFluor710, Streptavidin-Alexa Fluor 700 (eBioscience), FoxP3-Alexa Fluor 488, CD25-Brilliant Violet 421 (BioLegend), CD39-biotin, CD127-PE, CD4-APCVio770, CD3-APCVio770 (Miltenyi biotec), Streptavidin-ECD, CD45RO-ECD (Beckman Coulter). LIVE/DEAD fixable aqua staining kit (Life technologies) was used to discriminate live and dead cells. For intracellular staining, FoxP3 buffer set (eBioscience) was used. Cell acquisition was performed by an LSR II (Becton Dickinson) and analyses were performed using FlowJo software.
Brezar V., Hani L., Surenaud M., Hubert A., Lacabaratz C., Lelièvre J.D., Levy Y, & Seddiki N. (2017). Negative modulation of suppressive HIV-specific regulatory T cells by IL-2 adjuvanted therapeutic vaccine. PLoS Pathogens, 13(7), e1006489.
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5

Multiparametric Flow Cytometry Assay

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All staining experiments were performed at 4°C for 30 minutes. Antibodies used were CD3-PerCPCy5.5, CD8-APCCy7, CD25-APC, CD134-PE, TNF-α-PECy7, CD154-APC ((Becton Dickinson (BD) Biosciences)), CD4-Alexa Fluor 700, IFN-γ-eFluor450, IL2-PerCPeFluor710, Streptavidin-Alexa Fluor 700 (eBioscience), FoxP3-Alexa Fluor 488, CD25-Brilliant Violet 421 (BioLegend), CD39-biotin, CD127-PE (Miltenyi biotec), Streptavidin-ECD, CD45RO-ECD (Beckman Coulter). LIVE/DEAD fixable aqua staining kit (Life technologies) was used to discriminate live and dead cells. For intracellular staining, FoxP3 buffer set (eBioscience) was used.
Brezar V., Ruffin N., Richert L., Surenaud M., Lacabaratz C., Palucka K., Thiébaut R., Banchereau J., Levy Y, & Seddiki N. (2015). Decreased HIV-Specific T-Regulatory Responses Are Associated with Effective DC-Vaccine Induced Immunity. PLoS Pathogens, 11(3), e1004752.
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6

Flow Cytometry Analysis of CAR T-Cells

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Cells were resuspended in FACS staining buffer (PBS + 3% fetal bovine serum) using the following antibodies: CD3 (clone OKT3, eBiosciences), PD-1 (clone J105, eBiosciences), TIM3 (clone F38–2E2, BioLegend), Lag3 (clone 3DS223H, Invitrogen). CARs were detected using a PE or APC-conjugated CD22, CD19 or CD33 protein. All changes in overall tumor or T cell counts reflect changes in absolute cell counts, which were determined using CountBright absolute counting beads (ThermoFisher). Cell viability was established using Live/Dead Aqua fixable staining kit (ThermoFisher), and data were acquired on an LSRII Fortessa Cytometer (BD). All data analysis was performed using FlowJo 9.0 or 10 software (FlowJo, LLC). See Supplementary Figure 6 for gating strategy.
Singh N., Frey N.V., Engels B., Barrett D.M., Shestova O., Ravikumar P., Cummins K.D., Lee Y.G., Pajarillo R., Chun I., Shyu A., Highfill S.L., Price A., Zhao L., Peng L., Granda B., Ramones M., Maggie Lu X., Christian D.A., Perazzelli J., Lacey S.F., Roy N.H., Burkhardt J.K., Colomb F., Damra M., Abdel-Mohsen M., Liu T., Liu D., Standley D.M., Young R.M., Brogdon J.L., Grupp S.A., June C.H., Maude S.L., Gill S, & Ruella M. (2021). Antigen-independent activation enhances the efficacy of 41BB co-stimulated CD22 CAR T cells. Nature medicine, 27(5), 842-850.
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7

Quantifying Tumor-Infiltrating T Cells

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Cells were resuspended in FACS staining buffer (PBS + 3% fetal bovine serum) using the following antibodies: CD3 (clone OKT3, eBiosciences), CD4 (clone OKT4, eBiosciences), CD8 (clone SK1, BD), PD-1 (clone J105, eBiosciences), TIM3 (clones F38–2E2, BioLegend), Tbet (clone 4B10, eBiosciences). CD19 CAR was detected using an anti-idiotype antibody provided by Novartis Pharmaceuticals. All changes in overall tumor or T cell counts reflect changes in absolute cell counts, which were determined using CountBright absolute counting beads (ThermoFisher). Cell viability was established using Live/Dead Aqua fixable staining kit (ThermoFisher), and data were acquired on an LSRII Fortessa Cytometer (BD). All data analysis was performed using FlowJo 9.0 software (FlowJo, LLC).
Singh N., Lee Y.G., Shestova O., Ravikumar P., Hayer K.E., Hong S.J., Lu X.M., Pajarillo R., Agarwal S., Kuramitsu S., Orlando E.J., Mueller K.T., Good C.R., Berger S.L., Shalem O., Weitzman M.D., Frey N.V., Maude S.L., Grupp S.A., June C.H., Gill S, & Ruella M. (2020). Impaired death receptor signaling in leukemia causes antigen-independent resistance by inducing CAR T cell dysfunction. Cancer discovery, 10(4), 552-567.
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