Peripheral blood mononuclear cells (PBMC) were isolated from 9 ml EDTA tubes by gradient centrifugation in cell separation media (Lympholyte®-H, Cedarlane Labs, Burlington, Ontario). Cells were then stained with fluorochrome-conjugated monoclonal antibodies (mAb) (Supp. Table 2 ) using a 6 to 8-color immunophenotyping panel (Supp. Table 3 ) and analyzed by flow cytometry. Discrimination of live and dead cells was performed according to the manufacturer´s protocols (LIVE/DEAD® Fixable Aqua Dead Cell Staining Kit; Life Technologies, Oregon, USA). Total NK cells were identified as CD56posCD3neg cells and NK cell subsets as CD56brightCD16neg, CD56brightCD16low and CD56dimCD16high. Positive cells for a specific marker were determined as the percentage of positively stained cells minus the number of cells stained with an isotype-matched negative control antibody. Acquisition was performed either on a FACS Canto II or LSRII (BD Biosciences, San Jose, CA, USA). Flow cytometry analyses were performed with FACSDiva software v6.0 (BD Biosciences) and FlowJo vX (Tree Star, San Carlos, CA, USA).
Live dead fixable aqua dead cell staining kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The LIVE/DEAD Fixable Aqua dead cell staining kit is a fluorescent staining reagent used for the detection of dead cells in flow cytometry applications. The kit contains a cell-impermeant dye that binds to cellular proteins, allowing the identification of dead cells in a sample.
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Lab products found in correlation
Zenon technology, by Thermo Fisher Scientific (5 mentions)
Saponin, by Merck Group (4 mentions)
Anti cd107a af488 antibody, by BioLegend (4 mentions)
Anti cd3 pe cf594, by BD (4 mentions)
Anti ifn γ bv 421, by BioLegend (4 mentions)
Brefeldin a, by BD (4 mentions)
Monensin, by BD (4 mentions)
Anti cd56 apc, by BD (4 mentions)
Diva software, by BD (3 mentions)
Aria 2 flow cytometer, by BD (3 mentions)
Cd90.2 magnetic beads, by Miltenyi Biotec (3 mentions)
Cytofix cytoperm kit, by BD (3 mentions)
Ebioscience cell stimulation cocktail plus protein transport inhibitor, by Thermo Fisher Scientific (3 mentions)
Facscanto 2, by BD (2 mentions)
Cytoflex s, by Beckman Coulter (2 mentions)
Cytexpert software, by Beckman Coulter (2 mentions)
Fortessa, by BD (2 mentions)
Facsdiva software v6, by BD (1 mentions)
Lympholyte h, by Cedarlane (1 mentions)
Anti cd8, by Thermo Fisher Scientific (1 mentions)
21 protocols using live dead fixable aqua dead cell staining kit
1
Identification of NK Cell Subsets by Flow Cytometry
2
Comprehensive Immune Cell Profiling
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Isolated cells from spleens, joint footpads, and pLN were first stained with a Live/Dead fixable aqua dead cell staining kit (1:400) (Life Technologies) for 20 min. Cell were then washed and resuspended in 50 μl blocking buffer (1% rat and hamster serum in PBS). Staining was performed using anti-CD45 (BD Pharmingen), anti-CD4 (BD Pharmingen), anti-CD8 (eBioscience), anti-CD25 (eBioscience), anti-CD11b (eBioscience), anti-Ly6C (eBioscience), and anti-Ly6G (BioLegend) antibodies for 20 min. Stained cells were then washed with PBS and fixed using IC fixation buffer (eBioscience). Intracellular staining of anti-Foxp3 (eBioscience) was done according to the manufacturer's instructions. Data acquisition and analyses were done as described above.
3
Multicolor Flow Cytometry of Neutrophils
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Flow cytometry was performed on the Attune NxT Flow Cytometer (Life Technologies, CA, USA). Cells were first gated for doublet exclusion [forward scatter height (FSC-H) vs. forward scatter area (FSC-A)] followed by side scatter height (SSC-H) vs. FSC-H gating. Cell viability was checked by negative gating of cells stained with “Live/Dead Fixable Aqua Dead Cell Staining Kit” (Life Technologies, CA, USA). Neutrophils were analyzed by detecting the surface antigens with the following antibodies: Ly6G (clone 1A-8, BD Biosciences, CA, USA), CD11b (clone M1/70, Biolegend, CA, USA), CD54 (clone 3E2), and CD62L (clone MEL-14) from BD Pharmingen, CA, USA; or total antigens (surface and intracellular) by antibodies: CXCR4 (clone L276F12) and CXCR2 (clone SA045E1) from Biolegend, CA, USA; or by intracellular labeling with antibodies: Arg-1 (clone A1exF5) and inducible nitric oxide synthase (iNOS) (clone CXNFT) from Invitrogen, MA, USA. Cells were fixed with 1% paraformaldehyde and permeabilized with Saponin buffer [0.1% Saponin (w/v), 0.1% bovine serum albumin (BSA), 0.01 M HEPES, and 0.1% sodium azide in phosphate-buffered saline (PBS)] prior to the intracellular labeling as described previously (18 (link)).
4
Flow Cytometry Analysis of Cardiac Microtissues
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Flow cytometry was conducted in accordance with our previous study with modifications24 (link). Cardiac microtissues were dissociated by incubation with Accumax and stained with one or a combination of the following surface markers: anti-PDGFRβ conjugated with phycoerythrin (PE), clone 28d4, 1:100 (BD, Franklin Lakes, NJ, USA), and anti-VE-cadherin conjugated with allophycocyanin (APC), clone 55-7h1, 1:100 (BD). To eliminate dead cells, cells were stained with the LIVE/DEAD fixable Aqua dead cell staining kit (Thermo Fisher). For cell surface markers, staining was carried out in PBS with 5% FBS. For intracellular proteins, staining was carried out in cells fixed with 4% paraformaldehyde (PFA) in PBS. Cells were stained with the anti-cardiac isoform of troponin T (cTnT) (clone 13-11) (Thermo Fisher) labelled with APC using Zenon technology (Thermo Fisher) (1:50). The staining was performed in PBS with 5% FBS and 0.75% saponin (Sigma). The stained cells were analysed by a BD FACS Aria II (BD) or CytoFLEX S (Beckman Coulter, Brea, CA, USA). Data were collected from at least 10,000 events. Data were analysed with DIVA software (BD) or CytExpert software (Beckman Coulter).
5
Flow Cytometry Analysis of Cardiac Cells
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Flow cytometry was conducted in accordance with our previous study with modifications.1 Differentiated cardiovascular cells and cardiac tissue sheets were dissociated by incubation with Accutase and stained with one or a combination of the following surface markers: anti-PDGFRβ conjugated with phycoerythrin (PE), clone 28d4, 1:100 (BD, Franklin Lakes, NJ, USA) for MCs, and anti-VE-cadherin conjugated with phycoerythrin (PE), clone 55-7h1, 1:100 (BD) for ECs. To eliminate dead cells, cells were stained with the LIVE/DEAD fixable Aqua dead cell staining kit (Thermo Fisher). For cell surface markers, staining was carried out in PBS with 5% FBS. For intracellular proteins, staining was carried out in cells fixed with 4% paraformaldehyde (PFA) in PBS. Cells were stained with the anti-cardiac isoform of troponin T (cTnT) (clone 13-11) (Thermo Fisher) labelled with APC using Zenon technology (Thermo Fisher) (1:50) for CMs. The staining was performed in PBS with 5% FBS and 0.75% saponin (Nacalai Tesque). The stained cells were analyzed by CytoFLEX S (Beckman Coulter, Brea, CA, USA). Data was collected from at least 10,000 events. Data was analyzed with CytExpert software (Beckman Coulter). The percentage of CMs, ECs and MCs in cardiac tissue sheets used in the present study was as follows: CM, 35.53±10.47 %, EC, 1.86±0.93 %, MC, 37.43±7.07 % (n=6).
6
Flow Cytometry Analysis of Cardiomyocytes
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On d(9), we dissociated the cells using Accumax (Innovative Cell Technologies, San Diego, CA) and stained them with the cell surface markers listed in S1 Table . For cell surface markers, staining was carried out in PBS with 5% FCS. To eliminate dead cells, cells were stained with 4’,6-diamidino-2- phenylindole (DAPI) for surface marker staining or with the LIVE/ DEAD fixable Aqua Dead Cell Staining Kit (Thermo Fisher Scientific) for intracellular staining. For intracellular proteins, staining was carried out on cells fixed with 4% paraformaldehyde (PFA) in PBS. Cells were stained with the anti-cardiac isoform of Troponin T (TNNT2) (clone 13211, Thermo Fisher scientific) labeled with Alexa-488 using Zenon technology (Thermo Fisher Scientific). The staining was performed in PBS with 5% FCS and 0.75% Saponin (Sigma-Aldrich, St. Louis, MO). Stained cells were analyzed on an AriaII flow cytometer (Becton Dickinson, Franklin Lakes, NJ). After the selection of FSC/SSC gate, we additionally eliminated the doublets by SSC-W/SSC-H gate and FSC-W/FSC-H gate. Data were collected from at least 10,000 events.
7
Human iPSC Differentiation and Flow Cytometry
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During hiPSC differentiation and CTS generation, cells were dissociated by incubation with Accumax and stained with anti-PDGFRα antibody conjugated with APC (1:40, R&D) or anti-CD90 conjugated with APC (clone 5E10, 1:200, BioLegend, San Diego, USA). To eliminate dead cells, cells were stained with LIVE/DEAD fixable Aqua dead cell staining kit (Thermo Fisher Scientific). Cell surface marker staining was performed in PBS with 5% FBS. For intracellular proteins, staining was performed on cells fixed with 4% paraformaldehyde (PFA) in PBS. Cells were stained with the anti-cardiac isoform of Troponin-T (cTnT, clone 13211, 1:150 Thermo Fisher Scientific) labeled with Alexa-488 using Zenon technology (Thermo Fisher Scientific). The staining was performed in PBS with 5% FBS and 0.75% saponin (Sigma-Aldrich, St. Louis, USA). Stained cells were analyzed on an Aria II flow cytometer (BD, Franklin Lakes, USA). Data were collected from at least 5000 events and analyzed with DIVA software (BD).
8
Multiparametric Flow Cytometry Analysis
9
Apoptosis Assay for Multiple Myeloma
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MM cells (1 × 106) were treated with Pom for 5 days and then stained with the LIVE/DEAD Fixable Aqua Dead Cell Staining Kit (no. L34957, Thermo Fisher Scientific) and annexin V–phycoerythrin conjugate (no. 640908, BioLegend), according to the manufacturer’s instruction. Cells were analyzed in BD FACSCanto II (BD Biosciences) using the FACSDiva software (BD Biosciences).
10
Evaluating NK Cell Activity Assay
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The ADCC assay for measuring intracellular NK cell IFN-γ and CD107a expression was conducted and analysed with the gating strategy as previously described23 (link). Briefly, 96-well plates were coated overnight at 4 °C with A/California/04/09 HA protein (1 μg/ml) and chimeric cH9/1 HA protein (1 μg/ml). The plates were then washed with PBS and incubated with heat-inactivated sera (prediluted 1:10) for 2 h at 37 °C. Plates were then washed again with PBS and incubated with 105 CD16 176 v NK-92 cells per well (mycoplasma-free, human NK cell line expressing high affinity 176 V variant CD16 receptor) (Fox Chase Cancer Center, Philadelphia, PA, USA). As a negative control, NK-92 cells lacking the expression of CD16 were added to an additional well per sample. Cells were incubated with anti-CD107a-AF488 antibody (Biolegend, San Diego, CA, USA), Brefeldin A (5 μg/ml, BD) and monensin (5 μg/ml, BD) for 16 h at 37 °C. After incubation, the cells were stained with LIVE/DEAD Fixable Aqua dead cell staining kit (Invitrogen), anti-CD3-PE CF594 (BD) and anti-CD56-APC (BD) before intracellular staining with anti-IFN-γ-BV-421 (Biolegend). The cells were acquired on BD Fortessa. Data analysis was performed using FlowJo version 10 (treeStar).
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