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Aqua dead cell staining kit

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The Aqua dead cell staining kit is a fluorescent staining solution designed to identify and distinguish dead cells from live cells in various cellular applications. The kit contains a membrane-impermeable dye that selectively binds to dead cells, allowing for their detection and quantification using flow cytometry or other fluorescence-based methods.

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Lab products found in correlation

Irf4 3e4, by BD (2 mentions) Fc block 2.4g2, by BD (2 mentions) Brefeldin a, by Thermo Fisher Scientific (2 mentions) Sh800z cell sorter, by Sony (2 mentions) Foxp3 staining buffer set, by Thermo Fisher Scientific (2 mentions) Ionomycin, by Merck Group (2 mentions) Phorbol 12 myristate 13 acetate, by Merck Group (2 mentions) Fortessa, by BD (2 mentions) Foxp3 fixation permeabilization kit, by Thermo Fisher Scientific (2 mentions) Lsr fortessa cytometer, by BD (1 mentions) Anti mouse cd16 32 antibody, by BD (1 mentions) Cd4 rm4 5, by BD (1 mentions) Cd11b, by Miltenyi Biotec (1 mentions) Cd11c, by Miltenyi Biotec (1 mentions) Anti biotin and anti apc microbeads, by Miltenyi Biotec (1 mentions) Cd103, by BD (1 mentions) Murine il 36γ, by R&D Systems (1 mentions) Interleukin 2 (il 2), by Roche (1 mentions) Fix perm kit, by Thermo Fisher Scientific (1 mentions) Lsrfortessa flow cytometer, by BD (1 mentions)

8 protocols using aqua dead cell staining kit

1

Multiparameter Flow Cytometric Analysis

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Fluorescence dye labeled antibodies (Abs) specific for CD3 (145-2C11), CD4 (L3T4), CD25 (PC61.5), CD45 (30F11), CD45RB (C363.16A), CD11c (N418), TCRβ (H57-597), MHC-II (M5/114.15.2), Helios (22F6), FoxP3 (NRRF-30), IL-9 (RM9A4), pSMAD2/3 (O72-670), pSTAT5 (SRBCZX), pSTAT6 (18/P-Stat6), PU.1 (9G7), and IRF4 (3E4) were purchased from Becton Dickinson (BD), eBioscience, Biolegend and Cell Signaling Technology. Fc block (2.4G2) was purchased from BD. Dead cells were identified using the fixable Aqua dead cell staining kit (Invitrogen). Intracellular staining for Helios, IRF4 and Foxp3 was performed using a Foxp3 staining buffer set (eBioscience). Intracellular staining of IL-9 was performed after restimulation of cells with phorbol-12-myristate 13-acetate (Sigma), ionomycin (Sigma) and brefeldin A (eBioscience) for 4 hours. Stimulated cells were fixed and permeabilized, and then stained with Abs specific for IL-9. Detection of pSMAD2/3 was performed according to the BD Phosflow protocol. For intracellular detection of pSTAT5, pSTAT6 and PU.1, cells were fixed by 1.6% paraformaldehyde and incubated for 10 min at room temperature. Cells were then permeabilized by ice-cold methanol and stored at −80 °C before staining. Multi-parameter analysis was performed on a Fortessa (BD) and analyzed with FlowJo software (Tree Star). Cell sorting was performed using a SH800Z cell sorter (SONY).
Harusato A., Abo H., Le Ngo V., Yi S.W., Mitsutake K., Osuka S., Kohlmeier J.E., Li J.D., Gewirtz A.T., Nusrat A, & Denning T.L. (2017). IL-36γ signaling controls the induced regulatory T cell – TH9 cell balance via NFκB activation and STAT transcription factors. Mucosal immunology, 10(6), 1455-1467.
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2

Multiparameter Flow Cytometric Analysis

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Fluorescence dye labeled antibodies (Abs) specific for CD3 (145-2C11), CD4 (L3T4), CD25 (PC61.5), CD45 (30F11), CD45RB (C363.16A), CD11c (N418), TCRβ (H57-597), MHC-II (M5/114.15.2), Helios (22F6), FoxP3 (NRRF-30), IL-9 (RM9A4), pSMAD2/3 (O72-670), pSTAT5 (SRBCZX), pSTAT6 (18/P-Stat6), PU.1 (9G7), and IRF4 (3E4) were purchased from Becton Dickinson (BD), eBioscience, Biolegend and Cell Signaling Technology. Fc block (2.4G2) was purchased from BD. Dead cells were identified using the fixable Aqua dead cell staining kit (Invitrogen). Intracellular staining for Helios, IRF4 and Foxp3 was performed using a Foxp3 staining buffer set (eBioscience). Intracellular staining of IL-9 was performed after restimulation of cells with phorbol-12-myristate 13-acetate (Sigma), ionomycin (Sigma) and brefeldin A (eBioscience) for 4 hours. Stimulated cells were fixed and permeabilized, and then stained with Abs specific for IL-9. Detection of pSMAD2/3 was performed according to the BD Phosflow protocol. For intracellular detection of pSTAT5, pSTAT6 and PU.1, cells were fixed by 1.6% paraformaldehyde and incubated for 10 min at room temperature. Cells were then permeabilized by ice-cold methanol and stored at −80 °C before staining. Multi-parameter analysis was performed on a Fortessa (BD) and analyzed with FlowJo software (Tree Star). Cell sorting was performed using a SH800Z cell sorter (SONY).
Harusato A., Abo H., Le Ngo V., Yi S.W., Mitsutake K., Osuka S., Kohlmeier J.E., Li J.D., Gewirtz A.T., Nusrat A, & Denning T.L. (2017). IL-36γ signaling controls the induced regulatory T cell – TH9 cell balance via NFκB activation and STAT transcription factors. Mucosal immunology, 10(6), 1455-1467.
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3

Tumor Dissociation and Flow Cytometry

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BrM samples were collected after patients underwent craniotomy and surgical BrM resection. In the pSRS group, SRS was administered 1–10 days prior to craniotomy. All fresh samples (SOC and pSRS) for flow analysis were treated and acquired at Winship Cancer Institute. Samples were collected directly after resection into Phosphate Buffered Saline. The samples were then processed by getting cut into small pieces, digested with a MACS enzyme cocktail, and then homogenized using a MACS Dissociator. The digested tumor was washed then through a 70um filter to obtain a single cell suspension. Samples were then preserved in freezing media (FBS + 10% DMSO) at −80C.
Single cell suspensions from processed human tumor samples were stained with the antibodies listed Supplemental Table 5. Live/dead staining was done using fixable near-IR or aqua dead cell staining kit (Invitrogen). Cells were permed using the FOXP3 Fixation/Permeabilization kit (eBioscience) for 45 minutes with fixation/permeabilization buffer at 4C and stained with intracellular antibodies in permeabilization buffer for 30mins at 4C. Samples were acquired on a Symphony instrument and analyzed using FlowJo (v10).
Jansen C.S., Prabhu R.S., Pagadala M.S., Chappa P., Goyal S., Zhou C., Neill S.G., Prokhnevska N., Cardenas M., Hoang K.B., Zhong J., Torres M., Logan S., Olson J.J., Nduom E.K., del Balzo L., Patel K., Burri S.H., Asher A.L., Wilkinson S., Lake R., Higgins K.A., Patel P., Dhere V., Sowalsky A.G., Khan M.K., Kissick H, & Buchwald Z.S. (2023). Immune niches in brain metastases contain TCF1+ stem-like T cells, are associated with disease control and are modulated by preoperative SRS. Research Square.
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4

Flow Cytometric Characterization of Immune Cells

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After stimulation with or without CpG ODNs for 24 h, human PBMCs or mouse splenocytes were collected, pre-incubated with anti-mouse CD16/32 antibodies (BD Biosciences, San Jose, CA, USA), and the dead cells were excluded using an aqua dead cell staining kit (Invitrogen, San Diego, CA, USA). The cells were stained with the following antibodies: anti-human or anti-mouse CD45, CD3, CD19, CD80, and CD86 (BD Biosciences, San Jose, USA); incubated with the respective antibodies for 25 min at 4°C; and then washed twice. After performing FACS using LSRFortessa™ cytometer (BD Biosciences, San Jose, CA, USA), the results were analyzed using the FlowJo software (BD Biosciences, San Jose, CA, USA).
Li T., Wu J., Zhu S., Zang G., Li S., Lv X., Yue W., Qiao Y., Cui J., Shao Y., Zhang J., Liu Y.J, & Chen J. (2020). A Novel C Type CpG Oligodeoxynucleotide Exhibits Immunostimulatory Activity In Vitro and Enhances Antitumor Effect In Vivo. Frontiers in Pharmacology, 11, 8.
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5

Isolation and Flow Cytometry of Murine Intestinal Lamina Propria Cells

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The following antibodies were purchased from eBioscience: IFNγ (XMG1.2), CD90.1 (H1S51), CD69 (H1.2F3), CD45RB (C363.16A), CD45.1 (A20), Vα2 (B20.1), IL-17A (eBio17B7), CD8α (eBioT4/11.8), CD25 (PC61.5), CD3ε (eBio500A2), and RORγ(t)-PE (B2D). Antibodies purchased from BD Biosciences were: TCRβ (H57-597), Vβ5 (MR9-4), CCR6 (140706), IL-17A (TC11-18H10), Vα2 (B20.1), and CD4 (RM4-5). Dead cells were identified using the fixable Aqua dead cell staining kit (Invitrogen). The following biotin-conjugated antibodies (eBioscience) were used for negative selection in conjunction with anti-biotin and anti-APC microbeads (Miltenyi Biotec): CD8α (53-6.7), Ly-6G (RB6-8C5), F4/80 (BM8), TER-119 (TER-119), CD11b (M1/70), NK1.1 (PK136), CD11c (N418), CD19 (eBio1D3). Isolation of LP cells and flow cytometry was performed as previously described (18 (link)).
Geem D., Medina-Contreras O., McBride M., Newberry R.D., Koni P.A, & Denning T.L. (2014). Specific Microbiota-Induced Intestinal Th17 Differentiation Requires MHC II but not GALT and Mesenteric Lymph Nodes. Journal of immunology (Baltimore, Md. : 1950), 193(1), 431-438.
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6

Murine IL-36γ Cytokine Profiling

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Antibodies were from eBioscience except for CD45, CD103, CD4 (Becton Dickinson). Dead cells were identified using the Aqua dead cell staining kit (Invitrogen). Murine IL-36γ was from R&D. ELISAs for IL-36γ (antibodies-online.com) and IL-22, CXCL1, and CXCL2 (eBioscience) were performed following the manufacturer’s instructions.
Medina-Contreras O., Harusato A., Nishio H., Flannigan K.L., Ngo V., Leoni G., Neumann P.A., Geem D., Lili L.N., Ramadas R.A., Chassaing B., Gewirtz A.T., Kohlmeier J.E., Parkos C.A., Towne J.E., Nusrat A, & Denning T.L. (2015). IL-36 receptor promotes resolution of intestinal damage. Journal of immunology (Baltimore, Md. : 1950), 196(1), 34-38.
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7

Immune Cell Isolation and Analysis

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Isolated LP lymphocytes were resuspended in phosphate-buffered saline containing 5% fetal bovine serum. Live cells were identified using an Aqua Dead Cell Staining Kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions, and Fc receptors were blocked with the antibody anti-FcγRIII/II (2.4G2) for 15 minutes at 4°C. After incubation, the cells were stained at 4°C for 30 minutes with fluorescence-labeled antibodies. Samples then were washed 2 times in phosphate-buffered saline containing 5% fetal bovine serum and intracellular staining was performed using a Foxp3 fixation/permeabilization kit (eBioscience). Flow cytometric analysis was performed on a LSR II (BD Biosciences).
Geem D., Ngo V., Harusato A., Chassaing B., Gewirtz A.T., Newberry R.D, & Denning T.L. (2016). Contribution of Mesenteric Lymph Nodes and GALT to the Intestinal Foxp3+ Regulatory T-Cell Compartment. Cellular and Molecular Gastroenterology and Hepatology, 2(3), 274-280.e3.
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8

NK Cell Functional Evaluation

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Cryopreserved PBMCs were cultured overnight in RPMI 1640 media containing 10% FCS and 200 IU/ml IL-2 (Roche) and 1ng/ml IL-15 (Miltenyi) prior to functional analysis. NK cell degranulation and IFN-γ production assay was performed as previously described (13 (link)). To evaluate NK cell activity, PBMC were cultured for 5 hours with target cells in 96-well plates at a final E:T ratio of 5:1. CD107a was added at the beginning of co-culture, and GolgiStop (BD) was added to the co-culture one hour later. To assess antibody-dependent cellular cytotoxicity (ADCC), humanized anti-GD2 monoclonal antibody 3F8 (provided by Dr. Nai-Kong Cheung, MSKCC) was used at a concentration of 1μg/ml with a 1:20 dilution of sera collected from HCMV seropositive and seronegative healthy individuals. After surface marker staining, cells were fixed and permeabilized with a FIX&PERM kit (GAS004, Thermo Fisher) prior to intracellular IFN-γ staining. Dead cells were excluded from analysis using a fixable Aqua dead cell staining kit (ThermoFisher). Data were collected with a LSRFortessa flow cytometer (BD Bioscience) and analyzed using FlowJo (TreeStar).
Wu Z., Lau C.M., Sottile R., Le Luduec J.B., Panjwani M.K., Conaty P.M., Srpan K., Laib Sampaio K., Mertens T., Adler S.P., Hill A.B., Barker J.N., Cheung N.K., Sun J.C, & Hsu K.C. (2021). Human Cytomegalovirus Infection Promotes Expansion of a Functionally Superior Cytoplasmic CD3+ NK Cell Subset with a Bcl11b-Regulated T Cell Signature. The Journal of Immunology Author Choice, 207(10), 2534-2544.
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