The expression of cell markers and intracellular cytokines was analyzed by flow cytometry using the following antibodies and kits: anti-T-bet (REA102), anti-IFNγ (45–15), anti-TNFα (cA2) and anti-IL-2 (JES6-5H4), from Miltenyi Biotec; anti-CD25 (BC96), anti-FoxP3 (PCH101) and anti-PD-L1 (MIH1), from eBioscience and Annexin V Apoptosis Detection Kit with 7-AAD (Biolegend). Briefly, cells were washed with 0.5% BSA 2 mM EDTA in PBS (staining buffer), Fc receptors were blocked with 200 μg/mL IgG from human serum (Merck) and cells were stained with the fluorescence-labeled antibodies. T-bet and FoxP3 were detected by intracellular staining, using a FoxP3 staining buffer set (eBioscience, San Diego, CA) following the manufacturer’s instructions. Intracellular cytokines were stained as described previously (24 (link)), incubating cells with 10 μg/mL Brefeldin A (Merck) to stop Golgi protein transport for 4 hours before the FACS staining protocol. Data were acquired using a FACSCalibur flow cytometer (BD) and analyzed using FlowJo software (Tree Star, Ashland, OR). Gating strategy is shown in Supplementary Figure 1
Annexin 5 apoptosis detection kit with 7 aad
Manufactured by BioLegend
Sourced in United States
The Annexin V Apoptosis Detection Kit with 7-AAD is a lab equipment product that enables the detection and quantification of apoptosis in cell samples. Annexin V is used to identify apoptotic cells, while 7-AAD is used to distinguish between early apoptotic, late apoptotic, and necrotic cells. This kit provides the necessary reagents and protocols to perform these analyses.
Automatically generated - may contain errors
Lab products found in correlation
Brefeldin a, by Merck Group (1 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Canto 2 flow cytometer, by BD (1 mentions)
Xmarktm, by Bio-Rad (1 mentions)
Cell counting kit 8 cck, by Dojindo Laboratories (1 mentions)
Soluble αcd28 37, by BioXCell (1 mentions)
Celltrace violet ctv proliferation dye, by Thermo Fisher Scientific (1 mentions)
Murine il 2, by Thermo Fisher Scientific (1 mentions)
Cd8a t cell isolation kit, by Miltenyi Biotec (1 mentions)
7 aad, by BioLegend (1 mentions)
Facsverse flow cytometer, by BD (1 mentions)
Macsquant analyzer 10, by Miltenyi Biotec (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by BD (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Lsr 2, by BD (1 mentions)
11 protocols using annexin 5 apoptosis detection kit with 7 aad
1
Flow Cytometric Analysis of Immune Markers
2
Apoptosis Detection in LLC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
An Annexin V Apoptosis Detection Kit with 7-AAD (Biolegend) was used following the manufacturer's instructions. In brief, 2×105 LLC cells were cultured in 250 µl DMEM in 24-well culture plates. In addition, LLC cells were treated with or without recombinant Fgl2 (1 µg/ml or 5 µg/ml, R&D Systems) for 24 or 48 hours. Cells were harvested at the indicated time point and stained with FITC-Annexin V and 7AAD at a dilution of 1:20. The cells were incubated for 15 min at room temperature in the dark. Samples were acquired on a Canto II flow cytometer (BD Biosciences) and the data were analyzed with FlowJo software.
3
Annexin V-FITC and 7-AAD Apoptosis Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After transfection with pcDNA3.1-myc-his or pcDNA3.1-DCF1-myc-his for 48 h, the cells were harvested using trypsin (0.25% in PBS solution without EDTA), washed twice with 2%-BSA-PBS, and resuspended in 100 µl binding buffer combined with 5 µl Annexin V-FITC and gently mixed. Next, 10 µl 7-AAD was added to stain the cells according to the manufacturer's protocol for the Annexin V Apoptosis Detection kit with 7-AAD (BioLegend, San Diego, CA, USA). Samples were stained for 15 min at room temperature (RT), after which 400 µl binding buffer was added to prepare the samples for flow cytometry detection (Beckman Coulter, Inc.).
4
Evaluating Viability and Apoptosis of Cryopreserved MSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell proliferation and viability were assessed by using the CCK-8 Cell Counting kit (Dojindo, USA) and Annexin V apoptosis detection kit with 7-AAD (BioLegend, San Diego, CA). MSCs stored for 72 hours under hypothermic conditions from the eight different samples (including the controls) were harvested and analyzed. To determine the number of viable cells in the stocked vials, the cells were stained with Annexin V and 7-AAD antibodies according to the manufacturer’s instructions. The cells were resuspended in 500 μL of 1x staining buffer. Then, 5 μL of Annexin V and 7-AAD antibody were added to the binding buffer for 20–30 minutes at room temperature in the dark, prior to being analyzed by flow cytometry (BD Biosciences, USA).
To analyze the viability of the cells at various time points (0, 24, 48, 72 hrs), MSCs stored in CSF were seeded onto 96-well plates (3 × 103/well), and after 24 hours, CCK8 assay was performed. CCK-8 solution (10 uL) was added to each well, followed by incubation for 1 hour at 37 °C. The absorbance of CCK-8 was measured at 450 nm by using a microplate reader (x-MarkTM, Bio-Rad Laboratories, Inc, USA).
To analyze the viability of the cells at various time points (0, 24, 48, 72 hrs), MSCs stored in CSF were seeded onto 96-well plates (3 × 103/well), and after 24 hours, CCK8 assay was performed. CCK-8 solution (10 uL) was added to each well, followed by incubation for 1 hour at 37 °C. The absorbance of CCK-8 was measured at 450 nm by using a microplate reader (x-MarkTM, Bio-Rad Laboratories, Inc, USA).
5
In Vitro and In Vivo CD8+ T Cell Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD8+ T cells were purified from mouse spleen by MACS with the CD8a+ T cell isolation kit (Miltenyi Biotec, Auburn, CA, USA) and stained with cell trace violet (CTV) proliferation dye eBioscience (Thermo Fisher Scientific, Eugene, OR, USA). 0.2 x106 cells were stimulated with plate bound anti-CD3ε (145-2C11, 2 μg/mL) and soluble αCD28 (37.51, 1 μg/mL) (both from BioXcell, West Lebanon, NH, USA) in T cell media supplemented with murine IL-2 (10 ng/mL, PeproTech, Rocky Hill, NJ, USA). Proliferation was measured by dilution of the proliferation dye and apoptosis was assessed with an Annexin V apoptosis detection kit with 7-AAD from BioLegend (San Diego, CA, USA). For in vivo proliferation comparison, CD8+ T cells were purified from WT and Bcl3 KO P14 mouse spleen by MACS with the CD8a+ T cell isolation kit (Miltenyi Biotec, Auburn, CA, USA) and stained with cell trace violet (CTV) proliferation dye eBioscience (Thermo Fisher Scientific, Eugene, OR, USA). 1 x106 cells were adoptively transferred to congenic recipient mice. The next day, mice were infected with LCMV Armstrong and mesenteric lymph nodes were analyzed 4 days after infection.
6
Apoptosis Evaluation in Enriched T Lymphocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Enriched T lymphocyte cultures stimulated with ConA and treated with the IC50 dose of P. alata extract and polyphenols for 96 h were used to evaluate the frequency of cell death by apoptosis/necrosis. After culturing, the lymphocytes were washed with PBS 0.1 M (pH 7.4), centrifuged at 400× g for 5 min and stained with anti-CD4 and CD8 fluorochrome-conjugated monoclonal antibodies as described below. The cells were then washed with PBS 0.1 M (pH 7.4), centrifuged at 400× g for 5 min and stained with annexin-V and 7-Amino Actinomycin D (7-AAD) using an F.I.T.C. Annexin V apoptosis detection Kit with 7-AAD (Biolegend, San Diego, CA, USA) at the concentrations and with the procedures recommended by the manufacturer. Finally, 400 µL of Annexin V binding buffer was added to each tube, which was analyzed immediately via flow cytometry. We express the results as the percentages of viable, early apoptotic and late apoptotic/necrotic T cells. Figure S3 shows the representative dot plots demonstrating the quadrants that define the cell viability subsets.
7
Apoptosis and Necrosis Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded (5 × 104 per well in 24 wells microplates) and left to attach overnight before incubation with the material extracts for 24 h. Next day, to retain any cells lifting from the cultures due to apoptosis, culture supernatants were collected together with the fractions containing the PBS wash, and the cells still attached to the plastic harvested using trypsin-0.05% EDTA solution. The identification of apoptotic and necrotic cells was performed using Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend, San Diego, CA, USA). Briefly, cells were washed twice with cold Cell Staining Buffer, resuspended in Annexin V Binding Buffer before staining for 15 min with Annexin V-FITC reagent for early apoptotic cells' detection. To distinguish between the necrotic and apoptotic cells, the samples were also stained with 7-amino-actinomycin D (7-AAD) solution. Early apoptotic cells exclude 7-AAD, while late stage apoptotic and necrotic cells will stain positive for 7-AAD, which binds to DNA in the nucleus. Cells were analysed using a BD FACSVerse™ Flow Cytometer (BD Biosciences). The signal emitted by 7-AAD labelled cells was detected on the PerCP-Cy5.5 channel of the cytometer, while Annexin V positive cells were detected on the FITC channel.
8
Annexin V Apoptosis Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Apoptosis was assessed using the Annexin V Apoptosis Detection Kit with 7AAD (BioLegend, San Diego, CA). Forty-eight to seventy-two hours after radiation exposure, 2 x 105 cells were added in triplicate into a 96 well plate, rinsed with PBS, re-suspended in 10 µl of 1X annexin V binding buffer, and labeled with 1 µl of Annexin V for 15 min. 1 µl of 7AAD was then added for 5 min for dead cell discrimination. Finally, 180 µl was added to Annexin V binding buffer, following which samples were analyzed using the MACSQuant Analyzer 10 (Miltenyi Biotec Inc). Annexin V-/7AAD− cell population was considered healthy, Annexin V-/7AAD+ was indicative of necrotic cells, whereas the Annexin V+/7AAD− and Annexin V+/7AAD+ were representative of early and late apoptotic cells, respectively.
9
Investigating T Cell Proliferation and Apoptosis in OEC Co-Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-CD3/CD28 activated CD4 T cells were labeled with Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) (ThermoFisher) following the manufacturer's instructions and plated on 96-well cell-culture plates (2 × 105 cells/well) in complete RPMI media with or without OECs (8 T cell:1 OEC), that were previously treated for 48 h with or without MV130. Plates were incubated at 37°C and 5% CO2 for 7 days. Proliferation of CFSE-labeled CD4 T cells was monitored by flow cytometry (BD FACSCalibur). For apoptosis assays, anti-CD3/CD28 activated CD4 T cells in complete RPMI media on 96-well plates (2 × 105 cells/well) were incubated alone or with OECs (8 T cell:1 OEC) previously treated with or without MV130. After 48 h of culture at 37°C and 5% CO2, cell necrosis/apoptosis was assessed by flow cytometry (BD FACSCalibur) using Annexin V Apoptosis Detection Kit with 7-AAD (Biolegend).
10
Flow Cytometry Staining and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were stained for flow cytometry and included a pre-incubation step with unconjugated anti-CD16/32 (clone 93) to block Fc receptors as previously described (47 (link), 48 (link)). The antibody cocktails used for different sets of stains are listed in Supplementary Table 1
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!