For flow cytometric analysis, cells were stained with e506 LIVE/DEAD fixable dye (eBioscience) or 7-aminoactinomycin D (7-AAD) (BD Pharmigen) for 10 min on ice. Pelleted cells were incubated with Fc blocker (BioLegend) or normal mouse immunoglobulin G (IgG) (Life Technologies) for 10 min on ice to block the binding to Fc receptors. Extracellular antigens were stained for 20 min with antibodies on ice in a staining buffer. Cells were fixed and permeabilized with BD Cytofix/Cytoperm (for cytokine analysis) or eBioscience Transcription Factor Fix/Perm (for Bat3 or FoxP3 analysis) per the manufacturers’ instructions. Intracellular antigens were stained for 45 min on ice in the appropriate 1× perm/wash buffer before acquisition on a BD LSR II/Fortessa/Symphony flow cytometer (Becton Dickinson). The following antibodies were used (BioLegend unless otherwise specified): Bat3 (EPR9223, Abcam), anti-BrdU (3D4), FoxP3 (FJK-16, eBioscience), Granzyme B (QA16A02), IFN-γ (XMG1.2), IL-17A (Tc11–538 18H10.1), IL-10 (JES5–16E3), IL-2 (JES6–5H4), and TNF-α (MP6-XT22). Negative staining was determined using the appropriate isotype control antibody or Fluorescence Minus One control. Counting beads (BioLegend) were added to quantify absolute cell numbers. Results were acquired with the Diva software and analyzed using FlowJo software (Treestar).
7 aminoactinomycin d 7 aad
Manufactured by BD
Sourced in United States, Germany, France, United Kingdom, Italy, Denmark
7-aminoactinomycin D (7-AAD) is a fluorescent dye used in flow cytometry applications. It is a sensitive nucleic acid stain that binds to DNA. 7-AAD can be used to detect cell viability and to distinguish between live, apoptotic, and necrotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (28 mentions)
Annexin 5, by BD (27 mentions)
Facscalibur, by BD (25 mentions)
Facsdiva software, by BD (24 mentions)
Annexin 5 fitc, by BD (20 mentions)
7 aad, by BD (19 mentions)
Facscalibur flow cytometer, by BD (17 mentions)
Annexin 5 apc, by BD (16 mentions)
Annexin 5 pe, by BD (14 mentions)
Cellquest software, by BD (12 mentions)
Facscanto 2 flow cytometer, by BD (12 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (10 mentions)
Facsaria 2, by BD (9 mentions)
Lsrfortessa, by BD (8 mentions)
Facscanto, by BD (7 mentions)
Annexin 5 binding buffer, by BD (6 mentions)
Facsaria, by BD (6 mentions)
Trypsin edta, by Thermo Fisher Scientific (6 mentions)
Lsr 2 flow cytometer, by BD (5 mentions)
Facscanto flow cytometer, by BD (5 mentions)
411 protocols using 7 aminoactinomycin d 7 aad
1
Flow Cytometry Immunophenotyping Protocol
2
Cell Viability and Morphology Analysis
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Cell morphology after the various treatments was assessed using a bright-field Olympus BX43 microscope with the CellSens software (Version 3.2) (Olympus, Tokyo, Japan). Subsequently, cell number and viability were analyzed using a Cell Scepter electronic cell counter (EMD Millipore Corporation, Billerica, MA, USA) and the WST-1 assay (Roche Diagnostics, Taipei, Taiwan), respectively; moreover, the percentage of tBH-induced apoptotic cells was examined using 7-amino actinomycin D (7-AAD) (BD Pharmigen, Franklin Lakes, NJ, USA) staining and detected by a NucleoCounter ® NC-3000™ fluorescence image cytometer (ChemoMetec, Alleroed, Denmark). The filopodia lengths of the treated cells were manually quantified using the ImageJ software (Version 1.48) (National Institutes of Health, Bethesda, MD, USA).
3
Flow Cytometry Analysis of Mitochondrial Membrane Potential and Cell Death
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Cells treated with (Ra)-7 or DMSO control were centrifuged, washed once with PBS and stained with tetramethylrhodamine ethyl ester (TMRE) (cat#564696, BDBioscience) for 20 min at 37 °C, 7-amino-actinomycin D (7-AAD) (cat#51-68981E, BDPharmigen) or Annexin V-FITC (cat#556419, BDPharmigen) and propidium iodide (cat#P3566, Invitrogen) for 15 min in the dark at room temperature. Samples were then centrifuged, washed twice with PBS and analyzed by flow cytometry in a BD LSRFortessa. FACSDiva software was used for data collection and FlowJo software for data analysis. Gating strategies are shown in Supplementary Figure 8 .
Data presented in Fig.4b was calculated as follows:
Data presented in Fig.
4
Apoptosis Quantification by Flow Cytometry
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Cells were harvested with trypsin (Gibco, U.S.A.), washed with phosphate-buffered saline, and suspended in 1× binding buffer. Next, 5 µl AnnexinV-PE (BD Biosciences, U.S.A.) and 5 µl 7‐amino‐actinomycin D (7AAD) (BD Biosciences, U.S.A.) were added to the cell suspension, followed by a 15–20 min incubation at room temperature in the dark. About 400 µl of 1× binding buffer was then added to each tube. Fluorescence was detected by a flow cytometer (BD Biosciences, U.S.A.) and apoptosis was quantified using Flowjo software. The total apoptosis rate was determined by AnnexinV-PE and 7AAD assay Q2 (early apoptosis) + Q3 (late apoptosis).
5
Complement-Dependent Cytotoxicity Assay
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The complement-dependent cytotoxicity (CDC) assay was performed using fresh peripheral blood mononuclear cells (PBMCs) from the kidney donor. Briefly, 50 000 PBMCs were incubated with 10 μL of the patient serum and 10 μL of rabbit complement (Cat no. S7764) (Sigma-Aldrich, St. Louis, MO, USA) for 35 minutes at room temperature. Then, 5 μL of 7-aminoactinomycin D (7-AAD) (Cat no. 559925) (BD Biosciences, Franklin Lakes, NJ, USA) was added and incubated for another 10 minutes in the dark. The 7-AAD compound is a fluorescent intercalator that undergoes a fluorescence spectral shift upon association with DNA. The samples underwent fluorescence-activated cell sorting (FACS) using the BD FACSCanto II™ (BD Biosciences, Franklin Lakes, NJ, USA). The percentage of cells that underwent complement-mediated cell death was corrected by the negative control. A positive result was regarded as >10% of dead cells.
6
Immunomodulatory Effects of AuNPs on BMDCs and BMDMs
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The AuNPs exposed BMDMs and BMDCs were stimulated with 2 µg/mL LPS from E. coli, for 24 h at 37 °C with 5% CO2. The supernatant was harvested for cytokine immunoassay, and the cells were labelled with antibodies specific for CD11b (Ozyme, Saint Cyr, France, cat. no.: BLE101226) and CD11c (Ozyme, cat. no.: BLE117318), or CD11b (Ozyme, cat. no.: BLE101216) and F4/80 (Ozyme, cat. no.: BLE123152), cell surface markers of BMDCs and BMDMs, respectively, after Fc receptor blocking (BD Pharmingen, Claix, France, cat. no.: 553142) to reduce non-specific binding. To evaluate cellular activation, BMDCs and BMDMs were immunostained with anti-IAb-Ab (Ozyme, cat. no.: BLE116410) and anti-CD86 (Ozyme, cat. no.: BLE105008) antibodies. In both cases, only live cells were selected by staining with 7-Aminoactinomycin D (7AAD) (negative gating) (BD Biosciences, cat. no.: 559925) and analysed by flow cytometry using BD™ LSR II (BD Biosciences). The proportion of activated cells was quantified using FCS Express V6 (De Novo Software).
7
Cell Cycle Analysis by Flow Cytometry
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Cell cycle was analyzed by flow cytometry with an allophycocyanin (APC) BrdU Flow Kit according to the manufacturer's protocol (BD Biosciences, Mississauga, ON). Cells were labeled with bromodeoxyuridine (BrdU) for 2 hours at 37ºC. Cells were then incubated with the APCconjugated anti-BrdU antibody using a 1:100 dilution. Cells were additionally stained with 7amino-actinomycin D (7-AAD; BD Pharmingen) to determine cell cycle position. To stain cells with 7-AAD, cells were suspended in Dulbecco's phosphate buffered saline (DPBS) supplemented with 5 mM ethylenediaminetetraacedic acid (EDTA) and 0.5% bovine serum albumin (BSA) and then incubated with 7-AAD.
8
Autophagy Regulation in Bone Homeostasis
9
Apoptosis and Necrosis Quantification in U-87 Cells
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The induction of apoptosis and necrosis
of U-87 was measured 4 h after X-ray exposure by flow cytometry. X-ray-exposed
unstained and NT-PEO-Por*-stained U-87 were detached from dishes by
0.25% trypsin–EDTA, centrifuged at 1200 rpm for 5 min, and
resuspended in 100 μL of the annexin buffer (BD) and 5 μL
of Annexin V BV421 (BD), which binds to phosphatidylserin (PS) residues
and allows the identification of apoptotic cells. Cells were incubated
a 4 °C for 20 min, washed in the annexin buffer, and centrifuged.
The viability dye 7-aminoactinomycin D (7-AAD, BD) was added to cell
pellets to stain non-viable cells and recognize late-apoptotic fractions.
For fluorescence-activated cell sorting (FACS) characterization, data
were obtained using a FACSAria Fusion cell sorter, equipped with five
lasers, and analyzed with FACSDiva software (ver. 8.0, BD). At least
10 × 103 events were recorded for each condition.
Debris events were excluded from the analysis by morphological gating
(side scatter vs forward scatter dot plot).
of U-87 was measured 4 h after X-ray exposure by flow cytometry. X-ray-exposed
unstained and NT-PEO-Por*-stained U-87 were detached from dishes by
0.25% trypsin–EDTA, centrifuged at 1200 rpm for 5 min, and
resuspended in 100 μL of the annexin buffer (BD) and 5 μL
of Annexin V BV421 (BD), which binds to phosphatidylserin (PS) residues
and allows the identification of apoptotic cells. Cells were incubated
a 4 °C for 20 min, washed in the annexin buffer, and centrifuged.
The viability dye 7-aminoactinomycin D (7-AAD, BD) was added to cell
pellets to stain non-viable cells and recognize late-apoptotic fractions.
For fluorescence-activated cell sorting (FACS) characterization, data
were obtained using a FACSAria Fusion cell sorter, equipped with five
lasers, and analyzed with FACSDiva software (ver. 8.0, BD). At least
10 × 103 events were recorded for each condition.
Debris events were excluded from the analysis by morphological gating
(side scatter vs forward scatter dot plot).
10
Flow Cytometry Analysis of CAR.CD123 Expression
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The anti-human antibodies used for flow cytometry of HD and AML patient primary cells are listed in the Additional file 1 . In all analyses, the population of interest was gated based on forward vs. side scatter characteristics followed by singlet gating, and live cells were gated using 7-amino-actinomycin D (7-AAD) (BD Biosciences, Italy). The expression of CAR.CD123 on T and NK cells was detected by an anti-CD34 (cat. no FAB7227P, R&D Systems) and was evaluated over time, as indicated, in association with CD56- or CD3-specific mAbs and the isotype control. Flow cytometry analyses were performed on a FACSCanto II or LSRFortessa (BD Biosciences), and the data were analysed using FACSDiva software (BD Biosciences).
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