Mononuclear cell suspensions were incubated for 15 min with anti-CD16/32 to block Fc receptors and then with antibodies, anti-CD4 APC (RMA4-5, eBioscience), CD8 V450 (53.6.7, BD Bioscience), anti-CD11b APC-eFluor780 (M1/70, eBioscience), CD45 V500 (30F11, BD Bioscience), anti-CD19 FITC (1D3, BD Bioscience) and anti-TCR PerCP-Cy5.5 (H57, eBioscience) for 30 min on ice. LIVE/DEAD Fixable Cell Stain Kit (Life Technologies) was used to remove dead cells. All data were acquired using a digital flow cytometer (LSR II Fortessa, Becton Dickinson), and analysed with FlowJo software (Tree Star).
Live dead fixable cell stain kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Live/Dead Fixable cell stain kit is a fluorescent stain used to discriminate between live and dead cells in a sample. It is designed to label dead cells with a fluorescent dye, allowing for the identification and quantification of live and dead cells in flow cytometry or microscopy analysis.
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Lab products found in correlation
Foxp3 staining buffer kit, by Thermo Fisher Scientific (5 mentions)
Facsdiva software, by BD (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Anti cd19 fitc 1d3, by BD (1 mentions)
Lsrii fortessa, by BD (1 mentions)
Phosphate buffer saline (pbs), by Merck Group (1 mentions)
Anti cd86 pe cy7, by BD (1 mentions)
Anti cd11c, by BioLegend (1 mentions)
Lsrfortessa x 20 flow cytometer, by BD (1 mentions)
Lsm780 laser scanning, by Zeiss (1 mentions)
Zen 2010, by Zeiss (1 mentions)
F7378, by Merck Group (1 mentions)
Cytoflex s, by Beckman Coulter (1 mentions)
Thrombin, by Merck Group (1 mentions)
Annexin 5, by BioLegend (1 mentions)
C3c antibody, by Agilent Technologies (1 mentions)
Sabouraud glucose medium, by BD (1 mentions)
Amphotericin b deoxycholate, by Merck Group (1 mentions)
20 protocols using live dead fixable cell stain kit
1
Multiparameter Flow Cytometry Analysis
2
Multiparameter Immune Cell Profiling
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2 × 105 DC/mL were incubated in Phosphate Buffer Saline (PBS; Sigma-Aldrich, Merck) with the optimal dilution of anti-CD14 PE (MEM15; Exbio), anti-CD11c (3.9; Biolegend), anti-HLA-DR V500 (G46-6; BD Biosciences), anti-CD86 PE-cy7 (2331/FUN-1, B), and anti-CD40 Horizon 450 (5C3; BD Biosciences) antibodies for 20 min at 4°C. Cells were then washed twice with PBS and resuspended in 200 μL of 4% Formaldehyde-PBS. The Live/Dead Fixable Cell Stain Kit (Life Technology, Thermo Fisher Scientific) was added to the assay according to the manufacturer's instructions. A minimum of 50,000 events was acquired on a LSRFortessa™ X-20 flow cytometer (BD Biosciences) using the FACS Diva software (BD Biosciences). Data were analyzed using the FlowJo software (Tree Star). The gates strategy for one representative experiment was reported in Supplementary File 2 .
3
Flow Cytometric Quantification of Targeted Integration
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Reporterhigh expression was measured by flow cytometric analyses after 3–4 days post-electroporation and transduction using gates for multiplexed targeted integration set so that ‘AAV only’ samples (no nuclease) were less than 1% since previous data (not presented) have shown that after ~14 days in culture the frequency of reporter+ cells (from persistent episomal expression, random integration, and/or non-nuclease mediated HR) is generally less than 1%. The truncated NGFR receptor (tNGFR) where the cytoplasmic intracellular signaling domain is removed and is signaling incompetent, solely served the purpose of a reporter for targeted CD34+ HSPCs in indicated experiments (Bonini et al., 2003 (link)). Targeted integration of a tNGFR expression cassette was measured by flow cytometry of cells stained with APC-conjugated anti-human CD271 (NGFR) antibody (clone: ME20.4, BioLegend, San Diego, CA). For enriching of reporterhigh populations, cells were sorted on a FACS Aria II SORP using DAPI, PI (both Thermo Fisher, 1 µg/ml) or LIVE/DEAD Fixable Cell Stain Kit (Life Technologies) to discriminate live and dead cells according to manufacturer's instructions.
4
Immunofluorescence Staining Protocol
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Cells were fixed in 2% PFA, permeabilized in 2% saponin, and stained with antibodies as indicated. LIVE/DEAD Fixable Cell Stain Kit (Thermo Fisher Scientific) was used per the manufacturer’s instructions. Images were acquired using a Zeiss LSM780 Laser scanning confocal microscope and Zen2010 software (Carl Zeiss).
5
Caspase-1 Activity in MDM by Flow Cytometry
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The detection of caspase-1 activity in MDM FAM-FLICA® Caspase-1 Assay Kit (Immunochemistry Technologies) and flow cytometry, according to the manufacturer's instructions. Briefly, 2 x 105 MDM were stimulated according to the above-mentioned protocol, and then incubated with the fluorescent inhibitor probe FAM-YVAD-FMK for 1 h at 37°C, 5% CO2. The samples were then washed, incubated with Live/Dead Fixable Cell Stain Kit (Thermo Fisher Scientific), and analyzed by flow cytometry. The caspase-1 activity was expressed as percentage of FAM-FLICA positive (+) live cells.
6
Fetal Rat Skin Explant Viability
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For viability analysis, fetal rat skin explants were stretched and subsequently transferred in medium with fluorescein diacetate (25 µg mL−1, F7378, Sigma, USA) and ethidium bromide (10 µg mL−1) for 15 min at 37 °C. A positive control was kept in medium supplemented with 1% sodium azide for 2 h prior to staining. Imaging was directly performed after a brief washing step with fresh medium at the LSM 880 confocal microscope (Zeiss, Germany) with a 20× plan apochromatic objective and appropriate filter settings. The SEEs were transferred in trypsin-EDTA solution (0.05%, 25300062, Thermo Fisher Scientific, USA) and incubated for 10 min. Once the cell sheets started to break into pieces by smooth agitation, the SEEs were mechanically dissolved by pipetting up and down. The cells were then centrifuged and the pellet either resuspended in PBS with FBS (10%, 10270106, Thermo Fisher Scientific, USA) or in PBS with EtOH (35%) as positive control. The following viability assay was performed with the LIVE/DEAD™ Fixable Cell Stain Kit (L34963, Thermo Fisher Scientific, USA) according to the manufacturer’s protocol and analyzed by flow cytometry (Cytoflex S, Beckman Coulter, Brea, CA, USA).
7
Antifungal Susceptibility Testing Protocol
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All antibodies as well as annexin V were purchased from BioLegend (San Diego, CA, USA) except the C3c antibody, which was from Dako (Glostrup, Denmark), and the C5b-9 antibody, which was from Hycult (Uden, The Netherlands). The Live/Dead fixable cell stain kit was from Thermo Fisher Scientific (Waltham, MA, USA), and Sabouraud and TSB medium were from BD Diagnostic Systems (Franklin Lakes, NJ, USA). Liposomal amphotericin B (Ambisome) was purchased from Gilead (Foster City, CA, USA); amphotericin B deoxycholate, thrombin, and calcofluor white stain were from Sigma-Aldrich (St. Louis, MO, USA).
RPMI 1640 medium (R6504; Sigma-Aldrich) was supplemented with 19.8 g/L glucose and 34.5 g/L MOPS (morpholinepropanesulfonic acid) and adjusted to pH 7.0 with NaOH. Sabouraud glucose medium and agar were purchased from BD Diagnostic Systems (Heidelberg, Germany).
AmB-D and L-Amb were initially dissolved in dimethyl sulfoxide (DMSO). Stock solutions were further diluted in RPMI 1640 medium to the required concentrations. Medium controls always contained corresponding concentrations of DMSO.
RPMI 1640 medium (R6504; Sigma-Aldrich) was supplemented with 19.8 g/L glucose and 34.5 g/L MOPS (morpholinepropanesulfonic acid) and adjusted to pH 7.0 with NaOH. Sabouraud glucose medium and agar were purchased from BD Diagnostic Systems (Heidelberg, Germany).
AmB-D and L-Amb were initially dissolved in dimethyl sulfoxide (DMSO). Stock solutions were further diluted in RPMI 1640 medium to the required concentrations. Medium controls always contained corresponding concentrations of DMSO.
8
Multiparameter Flow Cytometry for Cell Phenotyping
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Cells were incubated with Fragment, crystallizable (Fc) block for 15–20 min, and then stained with primary antibodies (Table S1 ) for 20–30 min in buffer that contained Fc block. Stains with fluorescently labeled streptavidin were incubated for 20 min. Live cells were stained using a Live/Dead Fixable Cell Stain Kit (Thermo Fisher Scientific) or DAPI (Sigma). Intracellular staining was conducted using the eBioscience Transcription Factor staining kit. Cells were run on a FACSCantoII (BD Biosciences) and analyzed using FlowJo (FlowJo LLC). Lineage cocktails included antibodies to CD3e, CD4, CD5, CD8a, CD19, CD11b, CD11c, Gr-1, NK1.1, Ter119, and B220. “Modified” lineage cocktails included antibodies to CD3e, CD4, CD5, CD8a, CD19, CD11b, CD11c, Gr-1, NK1.1, Ter119, and F4/80.
9
Multicolor Flow Cytometric Analysis of BMMCs
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BMMCs or cell lines were stained using the Live/Dead Fixable Cell Stain Kit (Thermo Fisher Scientific, Massachusetts, USA) to exclude dead cells from the analysis. When analyzing plasma cells, BMMCs were immediately employed for flow cytometry staining without overnight resting in culture. After washing with flow cytometry (FACS) staining buffer, these cells were stained with the indicated fluorochrome-conjugated antibodies for 20 min at RT. For intracellular staining, surface-stained cells were fixed and permeabilized using an FoxP3 Staining Buffer Kit (Thermo Fisher Scientific, Massachusetts, USA) according to the manufacturer’s instructions. Multicolor flow cytometry was performed using an FACSLyric (BD Biosciences, New Jersey, USA) and the data was analyzed by FlowJo V.10 software (Treestar). The antibodies used for flow cytometry are detailed in online supplemental table 2 , and the gating strategies are summarized in online supplemental figures 3A, 4 and 8 .
10
Isolating and Analyzing Lymphocytes
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