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Zombie aquatm

Manufactured by BioLegend
Sourced in United States

Zombie AquaTM is a lab equipment product designed to facilitate cell culture and analysis. It serves as a specialized container for maintaining cells in an aquatic environment. The core function of Zombie AquaTM is to provide a controlled and optimized setting for culturing and observing cells.

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5 protocols using zombie aquatm

1

NK Cell Activation and Degranulation

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TLR activation of NK cells was analysed concerning induction IFN-gamma and TNF-alpha as well as CD107a degranulation in response to K562 target cells. Purified NK cells were pre-activated overnight with IL-2 and then cultured with and without Pam3CSK4, LPS-B5 and CpG-ODN-2216, respectively. After 16 h, NK cells were divided into either co-culture with K562 effector cells (effector to target ratio of 1:2) or kept in culture with medium alone. Next, FITC-conjugated CD107a (BD Biosciences, Heidelberg, Germany) was added. After 1 h, Golgi Stop (BD Biosciences) and BFA (Enzo Life Sciences GmbH) were added for additional 3 h. Then, cells were stained with Zombie AquaTM (BioLegend, London, UK) followed by staining with anti-CD3 (APC-Cy7-labelled), anti-CD56 (Brilliant Violet 421-labelled) and anti-CD16 (PerCP-labelled). After fixation and permeabilization, cells were stained intracellularly with anti-IFN-gamma (PE-labelled) and anti-TNF-alpha (PE-Cy7-labelled) (all BioLegend). Percent IFN-gamma-, TNF-alpha- and CD107a-positive CD56dimCD16pos, CD56dimCD16neg and CD56highCD16neg NK cells were measured before and after TLR stimulation as well as after co-culture of NK cells with K562 target cells in the presence and absence of TLR stimulation (Supplementary Figure 2).
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2

Sorting CD3+ T Cell Subsets

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To sort CD3, CD3+TCRαβ, and CD3+TCRαβ+ MDM, single-cell suspensions were obtained from the MDM cultures as described. The cells were incubated in Zombie AquaTM (BioLegend) for 20 min, at room temperature in the dark. Then, they were stained with BV421-label anti-TCRαβ mAb (clone IP26; BioLegend) and APC-labeled anti-CD3 mAb (clone UCHT1; BioLegend) for 30 min, at 4°C in the dark. Thereafter, the cells in suspension were sorted using a FACS Aria II (BD Biosciences) 85 μm nozzle using the following strategy: The dead cells were excluded by analyzing the negative cell region to the Zombie Aqua staining. The doublets were gated out by plotting the FSC area vs. the FSC height. Finally, the live CD3, CD3+ TCRαβ, and CD3+TCRαβ+ cells were individually sorted.
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3

Phenotypic Characterization of Human and Mouse Myeloid Cells

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We evaluated cell surface marker expressions on cultured human macrophages using monoclonal antibodies (mAbs) to CD80, CD86, CD11b, CD68, CD14, CD16, HLA-ABC, HLA-DR, CD1a, CD1b, CD1c, CD1d, the TCRβ chain, TCRγδ, CD3ε (epsilon chain), CCR4, CCR7, CXCR1, and TNF. Likewise, to evaluate mouse myeloid cells, we used mAbs to CD11b, CD3ε, TCRβ, TNFR1, and TNFR2. All the mAbs were provided by BioLegend (San Diego, CA, USA). The cells were stained for 30 min, at 4°C in the dark. Then, the cells were fixed by 2% p-formaldehyde in phosphate-buffered saline (PBS: 10 mM sodium phosphate, 0.15 M sodium chloride, pH 7.2). The cells used for Fluorescence Minus One (FMO) condition were stained and acquired in parallel to identify background levels of staining, dead cells were omitted by use of Zombie AquaTM (BioLegend) viability kit.
The data were collected by means of a FACS Aria II (BD Biosciences, San Jose, CA, USA) or FACS CyAn flow cytometer (Beckman Coulter, Inc. Brea, CA, USA) and analyzed by FlowJo v10.2 (FlowJo LLC, Inc, Ashland, OR, USA). In each case, 50,000 events were acquired per sample. A list of the antibodies clones used can be found in Table S1.
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4

Retinal Cell Viability and Characterization

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Cell viability was evaluated at the time of retinae cell dissociation. To ensure we obtain live cells after sorting, we labeled the cells with Zombie AquaTM (BioLegend, San Diego, CA, USA) a permeant dye to discriminate between live (negative for dye) and dead (positive for dye) cells. Live cells were treated with 1.0 μg of anti-CD16/CD32 per 1.0 × 106 cells in 100 μL to block FcγRII/III (clone 93; BioLegend), which minimizes non-specific binding of the primary antibodies and in turn inhibits endocytosis, phagocytosis and antigen presentation due to FcγR activation. The following primary antibodies were used to detect surface antigens by incubating the cells on ice for 30 min: anti-CD90.1 PerCP-Cy5.5 (Thy1.1, clone OX-7, BioLegend, exhibits no cross-reactivity with CD90.2); anti-CD90.2 Alexa Fluor-700 (Thy1.2, clone 30-H12, BioLegend, exhibits no cross-reactivity with CD90.1); anti-CD48 PE-Cy7 (clone HM48-1, BioLegend, labels monocytes and microglia); anti-CD15 PE (clone MC-480, BioLegend, labels amacrine cells); and anti-CD57 (clone VC1.1, Sigma Aldrich, St. Louis, MO, USA also labels amacrine cells). Because the anti-CD57 antibody was unconjugated, a Brilliant violet 421-tagged secondary antibody (Life Technologies, Carlsbad, CA, USA) was used to allow for sorting.
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5

Cellular Checkpoint Molecule Expression

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Expression of cellular checkpoint molecules was assessed by flow cytometry. Leukocytes from blood were isolated using an aggregation and gravitational separation method (HetaSepTM, STEMCELLTM technologies, Vancouver, Canada) according to the manufacturer’s instructions and stored in BamBanker freezing medium (STEMCELLTM technologies). About 5 x106 leukocytes were later stained on ice for 30 min (staining buffer, BioLegend, San Diego, CA). After washing, cells were taken up in 50 μl staining buffer containing a dead cell detection reagent (Zombie aquaTM, BioLegend). Expressions were measured by flow cytometry (Cytoflex s, Beckman Coulter, Brea, CA) and analyzed using a DeNovo software (FCS Express 7, Pasadena, CA). Details on antibodies used, FACS-settings, and gating strategy are given in the S2 File.
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