Fixable aqua

Manufactured by Thermo Fisher Scientific

Sourced in United States

Fixable Aqua is a viability dye used in flow cytometry applications. It is designed to identify and exclude dead cells from analysis.

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Lab products found in correlation

14 protocols using fixable aqua

Multicolor Flow Cytometry Analysis

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Cells from freshly collected cloudy peritoneal effluents were acquired on an eight-colour FACSCanto II (BD Biosciences) and analyzed with FlowJo 10.1 (TreeStar), using monoclonal antibodies against CD3 (SK7) and CD15 (HI98 or HIM1) from BD Biosciences; and anti-CD14 (61D3) from eBioscience. Leukocyte populations were gated based on their appearance in side scatter and forward scatter area/height and exclusion of live/dead staining (fixable Aqua; Invitrogen).

Brook A.C., Jenkins R.H., Clayton A., Kift-Morgan A., Raby A.C., Shephard A.P., Mariotti B., Cuff S.M., Bazzoni F., Bowen T., Fraser D.J, & Eberl M. (2019). Neutrophil-derived miR-223 as local biomarker of bacterial peritonitis. Scientific Reports, 9, 10136.

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Characterization of Monocytes and γδ T-Cells

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Cells were acquired on a BD LSRFortessa flow cytometer (BD Biosciences, Wokingham, UK) and analysed with flowjo version 10 (TreeStar, Ashland, OR, USA). Single cells of interest were gated based on their appearance in side and forward scatter area/height, exclusion of live/dead staining (fixable Aqua; Invitrogen) and surface staining (CD3⁻ CD19⁻ CD14⁺ monocytes, Vδ2⁺ CD3⁺ γδ T‐cells). The following mAbs were used for surface labelling: anti‐CD3 (SP34‐2), anti‐CD64 (10·1) and anti‐TCR‐Vδ2 (B6) from BD Biosciences; anti‐TCR‐pan‐γδ (Immu510) and anti‐CD40 (mAB89) from Beckman Coulter, High Wycombe, UK; and anti‐CD14 (M5E2), anti‐CD86 (IT2·2) and anti‐HLA‐DR (L243) from Biolegend.

Raffray L., Burton R.J., Baker S.E., Morgan M.P, & Eberl M. (2019). Zoledronate rescues immunosuppressed monocytes in sepsis patients. Immunology, 159(1), 88-95.

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Macrophage Lysosomal Enzyme Assay

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Human M1 macrophages were detached using 2 mM EDTA for 45 minutes and washed in PBS. Cells were pulsed with 240 μM C_₁₂FDG for 30 minutes and 200 μg/mL DQ-BSA (Thermo Fisher Scientific) for 15 minutes at 37°C, with Fixable Aqua (Invitrogen, Thermo Fisher Scientific) added in the final 10 minutes. Cells were washed before flow cytometry (BD Fortessa).
Human M1 macrophages were treated with TbAd (20 μM) and lalistat-2 (100 μM) in triplicate wells containing 3 × 10^⁵ cells/well in a 24-well plate and then lysed using water containing 0.5% Triton X-100. Lysate (10 μL) was transferred onto a 96-well plate with 30 μL buffer containing 4-propyl-8-methyl-7-hydroxycoumarin (P-PMHC) (Cayman Chemical), an LAL substrate, incubated at 37°C for 3 hours, and quenched with 200 μL of 50% methanol. Lysate (150 μL) in a black flat-bottomed, 96-well plate was read (BioTek, Synergy H1 fluorimeter) at 360/460 nm excitation/emission wavelengths.

Bedard M., van der Niet S., Bernard E.M., Babunovic G., Cheng T.Y., Aylan B., Grootemaat A.E., Raman S., Botella L., Ishikawa E., O’Sullivan M.P., O’Leary S., Mayfield J.A., Buter J., Minnaard A.J., Fortune S.M., Murphy L.O., Ory D.S., Keane J., Yamasaki S., Gutierrez M.G., van der Wel N, & Moody D.B. (2023). A terpene nucleoside from M. tuberculosis induces lysosomal lipid storage in foamy macrophages. The Journal of Clinical Investigation, 133(6), e161944.

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Flow Cytometry Analysis of Virus-Infected Cells

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Flow cytometry was performed using direct fluorescence-conjugates and their respective controls. Phycoerythrin (PE)-labeled anti-human CD46 antibody and PE-labeled IgG1 isotype control were from eBioscience. CD117-PeCy7 and CD33-BV421 conjugates were from BD Biosciences. Dead cells were excluded using either 7-aminoactinomycin D (BioLegend) or using Fixable Aqua (Invitrogen; Thermo Fisher Scientific) following extracellular staining according to the manufacturer's instructions. Cells employed in the virus infection experiments were fixed using cytofix buffer (BD Biosciences). Acquisition was done using a Canto-II (BD Biosciences) or an Attune NxT (Life Technologies) cytometer. Viable, single cells were used for further analysis as depicted in Fig. S1 apart from tetramethylrhodamine ethyl ester (TMRE) staining where only subcellular debris was excluded.

Maurer S., Salih H.R., Smirnow I., Lauer U.M, & Berchtold S. (2019). Suicide gene-armed measles vaccine virus for the treatment of AML. International Journal of Oncology, 55(2), 347-358.

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Multicolor Flow Cytometry Panel

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Burfeind K.G., Zhu X., Norgard M.A., Levasseur P.R., Huisman C., Michaelis K.A., Olson B, & Marks D.L. (2020). Microglia in the hypothalamus respond to tumor‐derived factors and are protective against cachexia during pancreatic cancer. Glia, 68(7), 1479-1494.

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Characterization of Peritoneal T-Cell Subsets

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Cells were acquired on an eight-color FACSCanto II (BD Biosciences) and analyzed with FlowJo 10.1 (TreeStar), using monoclonal antibodies against CD3 (SK7), CD69 (FN50), CCR4 (1G1), CCR5 (2D7) and CCR6 (11A9) from BD Biosciences; anti-TCR-Vγ9 (Immu360) from Beckman Coulter; anti-CD161 (HP-3G10), CCR2 (K036C2) and anti-TCR-Vα7.2 (3C10) from Biolegend; together with appropriate isotype controls. Anti-mouse antibodies reactive beads were used to set compensation (Life Technologies). Intracellular cytokines were detected using anti-IFN-γ (B27; Biolegend) and anti-TNF-α (188; Beckman Coulter). For detection of intracellular cytokines, 10 µg/ml brefeldin A (Sigma) was added to cultures 5 hours prior to harvesting. Leukocyte populations were gated based on their appearance in side scatter and forward scatter area/height and exclusion of live/dead staining (fixable Aqua; Invitrogen). Unless stated otherwise, peritoneal γδ T-cells were defined as Vγ9⁺ CD3⁺ lymphocytes. Peritoneal MAIT cells were defined as Vα7.2⁺ CD161⁺ CD3⁺ lymphocytes; control stainings using MR1 tetramers as reference confirmed the validity of this approach (data not shown).

Liuzzi A.R., Kift-Morgan A., Lopez-Anton M., Friberg I.M., Zhang J., Brook A.C., Roberts G.W., Donovan K.L., Colmont C.S., Toleman M.A., Bowen T., Johnson D.W., Topley N., Moser B., Fraser D.J, & Eberl M. (2016). Unconventional Human T Cells Accumulate at the Site of Infection in Response to Microbial Ligands and Induce Local Tissue Remodeling. The Journal of Immunology Author Choice, 197(6), 2195-2207.

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Multicolor Flow Cytometry Analysis

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Cells were acquired on an eight-color FACSCanto II (BD Biosciences) and analyzed with FlowJo (TreeStar). Single cells of interest were gated based on their appearance in side and forward scatter area/height, exclusion of live/dead staining (fixable Aqua; Invitrogen) and surface staining. Apoptotic cells were identified using annexin-V (BD Biosciences).

Davey M.S., Morgan M.P., Liuzzi A.R., Tyler C.J., Khan M.W., Szakmany T., Hall J.E., Moser B, & Eberl M. (2014). Microbe-specific unconventional T-cells induce human neutrophil differentiation into antigen cross-presenting cells. Journal of immunology (Baltimore, Md. : 1950), 193(7), 3704-3716.

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Comprehensive Immune Cell Profiling

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All antibodies were purchased from BioLegend, except for Live/Dead, which was purchased from Invitrogen (Fixable Aqua, used at 1:200 dilution). The following anti-mouse antibodies were used, with clone, fluorophore, and dilution indicated in parenthesis: CD3 (17A2, PE, 1:100), CD3 (17A2, APC/Cy7, 1:400), CD4 (RM4-5, APC, 1:100), CD8 (53–6.7, APC/Cy7, 1:800), CD11b (M1/70, APC, 1:800), CD11b (M1/70, FITC, 1:200), CD19 (6D5,BV650, 1:33), CD45 (30-F11, PerCP/Cy5.5, 1:400), CD45 (30-F11, APC/Cy7, 1:400), CX3CR1 (SAO11F11, PE/Cy7, 1:100), Dec205 (NLDC-145, APC,1:100), CD115 (AFS98, APC/CY7, 1:400), Ly6C (HK1.4, PerCP, 1:100), Ly6G (1A8, PE/Cy7, 1:800), NK1.1 (PK136, BV785, 1:800), CCR2 (SA203G11, AF647, 1:100).

Burfeind K.G., Zhu X., Norgard M.A., Levasseur P.R., Huisman C., Buenafe A.C., Olson B., Michaelis K.A., Torres E.R., Jeng S., McWeeney S., Raber J, & Marks D.L. (2020). Circulating myeloid cells invade the central nervous system to mediate cachexia during pancreatic cancer. eLife, 9, e54095.

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Multiparameter Flow Cytometry of Immune Cells

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All antibodies were purchased from BioLegend, except for Live/Dead, which was purchased from Invitrogen (Fixable Aqua, used at 1:200 dilution). The following anti‐mouse antibodies were used, with clone, fluorophore, and dilution indicated in parenthesis: CD3 (17A2, PE, 1:100), CD11b (M1/70, FITC, 1:200), CD45 (30‐F11, PerCP/Cy5.5, 1:400), Ly6C (HK1.4, PerCP, 1:100), Ly6G (1A8, PE/Cy7, 1:800), F4/80 (BM8, APC, 1:100), F4/80 (BM8, BV785, 1:100), CD206 (MMR, APC, 1:100).

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Multiparametric Flow Cytometry Analysis

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Cells were acquired on an eight-colour FACSCanto II (BD Biosciences, Oxford, UK) and analysed with FlowJo (TreeStar, Ashland, OR, USA). Single cells of interest were gated based on their appearance in side and forward scatter area/height, exclusion of live/dead staining (fixable Aqua; Invitrogen) and surface staining. The following monoclonal antibodies (mAbs) were used for surface labelling: anti-CD3 (UCHT1), CD8 (HIT8a and SK1), CD16 (3G8), CD24 (ML5), CD44 (G44-26), GD2 (14.G2a) from BD Biosciences; anti-TCR-Vγ9 (Immu360) from Beckman Coulter, High Wycombe, UK; and anti-HLA-ABC (w6/32) from Biolegend, London, UK; together with appropriate isotype controls. Intracellular cytokines were detected using anti-IFN-γ mAbs (B27, BD Biosciences). Surface mobilisation of CD107a was detected by adding anti-CD107a (H4A3; BD Biosciences) mAbs and GolgiStop (BD Biosciences) to cultures for 5 h prior to flow cytometric analysis.

Chen H.C., Joalland N., Bridgeman J.S., Alchami F.S., Jarry U., Khan M.W., Piggott L., Shanneik Y., Li J., Herold M.J., Herrmann T., Price D.A., Gallimore A.M., Clarkson R.W., Scotet E., Moser B, & Eberl M. (2017). Synergistic targeting of breast cancer stem-like cells by human γδ T cells and CD8+ T cells. Immunology and Cell Biology, 95(7), 620-629.

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