After treatment with 0, 20, 40 or 60 µM rapamycin for 24 hours, U87-MG cells were stained with apoptotic indicators, Fluorescein isothiocyanate-conjugated Annexin V (AV) and Propidium Iodide (PI), according to the manufacturer's instructions (BD Biosciences, San Diego, CA). Cell staining was analyzed using a CytoFlex flow cytometer (Beckman Coulter, Indianapolis, IN). The average percent of double positive-stained cells in each rapamycin treatment group was quantified and compared to untreated controls.
Fluorescein isothiocyanate conjugated annexin 5
Manufactured by BD
Sourced in United States
Fluorescein isothiocyanate-conjugated annexin V is a fluorescently labeled protein used to detect the presence of phosphatidylserine on the outer cell membrane, a marker of apoptosis or cell death.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by BD (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Trypan blue, by Thermo Fisher Scientific (1 mentions)
Countess 2 automated cell counter, by Thermo Fisher Scientific (1 mentions)
Wnt7a, by R&D Systems (1 mentions)
Anti fas antibody, by BD (1 mentions)
Wnt3a, by R&D Systems (1 mentions)
Binding buffer, by BD (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Hyclone, by GE Healthcare (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
7 aminoactinomycin d, by BD (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
11 protocols using fluorescein isothiocyanate conjugated annexin 5
1
Rapamycin Induces Apoptosis in U87-MG Cells
2
Cell Viability and Apoptosis Assay
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Cells were digested by 0.25% trypsin (Gibco, 25200114) for a few minutes, and the digestion was stopped by adding complete DMEM medium. The cell-containing medium was thoroughly pipetted up and down several times to form single-cell suspensions. For viability evaluation, we mixed an equal volume of cell suspensions and 0.4% trypan blue solution (Gibco, 15250061) well and transferred them into chamber slides (Invitrogen, C10312). Cell viability was measured as the percentage of cells excluded by trypan blue using the Countess II Automated Cell Counter (Invitrogen, AMQAX1000). For apoptosis assay, the cells were incubated with fluorescein isothiocyanate–conjugated annexin V (BD Biosciences, 556547) and subjected to fluorescence-activated cell sorting analysis according to the manufacturer’s protocol.
3
Chondrogenesis and Apoptosis Induction in Murine Chondrocytes
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Mesenchymal cells were derived from the limb buds of ICR mouse embryos 11.5 days postcoitus and maintained as micromass cultures for induction of chondrogenesis as described previously
[8 (link)]. Mouse articular chondrocytes were isolated from knee cartilage obtained from postnatal day 5 mice
[25 (link)]. The articular cartilage was preincubated for 2 hours at 37°C with 0.2% trypsin and 0.2% type II collagenase and further digested with 0.2% type II collagenase for 90 minutes. On culture day 3, the cells were treated with recombinant interleukin 1β (IL-1β) (Calbiochem, San Diego, CA, USA), Wnt3a or Wnt7a (R&D Systems, Minneapolis, MN, USA) for 24 hours. Apoptosis was induced by treatment with an anti-Fas antibody (BD Biosciences, San Jose, CA, USA). Briefly, chondrocytes from articular cartilage of WT or Lrp5-/- mice were incubated in the presence or absence of IL-1β (1 ng/ml) for 24 hours, then exposed to the anti-Fas antibody and recombinant protein G for an additional 6 hours. Hamster immunoglobulin G2 was used as a control. The cells were stained with fluorescein isothiocyanate–conjugated annexin V (BD Biosciences), and apoptotic chondrocytes were quantified by fluorescence-activated cell sorting analysis.
[8 (link)]. Mouse articular chondrocytes were isolated from knee cartilage obtained from postnatal day 5 mice
[25 (link)]. The articular cartilage was preincubated for 2 hours at 37°C with 0.2% trypsin and 0.2% type II collagenase and further digested with 0.2% type II collagenase for 90 minutes. On culture day 3, the cells were treated with recombinant interleukin 1β (IL-1β) (Calbiochem, San Diego, CA, USA), Wnt3a or Wnt7a (R&D Systems, Minneapolis, MN, USA) for 24 hours. Apoptosis was induced by treatment with an anti-Fas antibody (BD Biosciences, San Jose, CA, USA). Briefly, chondrocytes from articular cartilage of WT or Lrp5-/- mice were incubated in the presence or absence of IL-1β (1 ng/ml) for 24 hours, then exposed to the anti-Fas antibody and recombinant protein G for an additional 6 hours. Hamster immunoglobulin G2 was used as a control. The cells were stained with fluorescein isothiocyanate–conjugated annexin V (BD Biosciences), and apoptotic chondrocytes were quantified by fluorescence-activated cell sorting analysis.
4
Annexin V Apoptosis Assay
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After treatment with the drug or VH, trypsinized cells were incubated with 5 μl of fluorescein isothiocyanate-conjugated annexin V (BD Biosciences) in 1 × binding buffer (BD Biosciences) for 15 min at RT, according to the manufacturer’s protocol. The stained cells were analyzed by flow cytometry on a FACS Calibur instrument. Values are expressed as the percentage of fluorescent cells relative to the total cell count.
5
Apoptosis Induction in Mouse HSCs
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mHSCs were cultured at 37°C with 5% CO2 in Dulbecco’s Modified Eagle Medium containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cells were harvested and stained with propidium iodide and fluorescein isothiocyanate-conjugated annexin V (BD Biosciences) to detect apoptosis, according to the manufacturer’s instructions. Target cells alone were used as controls. Hepatic NK cells from Tigit-/- and WT mice were co-cultured with mHSCs at an effector-to-target ratio of 5:1 for 24 or 48 h, and the percentage of apoptotic mHSCs was calculated.
6
Apoptosis Analysis with Annexin V
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For the analysis of apoptosis. Fluorescein isothiocyanate–conjugated annexin V (BD PharMingen) and propidium iodide were used as described (Cruse et al., 2010 (link)).
7
Murine LLC Cell Line Cultivation and Apoptosis Analysis
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The murine LLC cell line was purchased from the Cell Bank of the Shanghai Institute for Biological Sciences, Chinese Academy of Sciences. LLC cells were maintained in high-glucose Dulbecco's modified Eagle's medium (HyClone; GE Healthcare Life Sciences) supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml) and 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) in a 5% CO2 humidified atmosphere at 37°C.
Cells which had adhered to the base of the T-25 flask were dislodged by aspiration several times with culture medium. The supernatants were used to stimulate neutrophils for NETs as described subsequently. The LLC cells were resuspended in PBS buffer with 1% BSA (Sigma-Aldrich; Merck KGaA). Aliquots containing 1×105 cells in 100 µl buffer were stained with 10-µl propidium iodide (50 mg/ml) solution and with 5 µl fluorescein isothiocyanate-conjugated Annexin V (17.6 mg/ml; BD Biosciences) for 5 min at 37°C. Following staining, 400 µl PBS was added to the cells. Immediately, flow cytometry analysis was performed using a FACScan flow cytometer (BD Biosciences). All FACS data were analyzed using FlowJo v10 software (FlowJo LLC).
Cells which had adhered to the base of the T-25 flask were dislodged by aspiration several times with culture medium. The supernatants were used to stimulate neutrophils for NETs as described subsequently. The LLC cells were resuspended in PBS buffer with 1% BSA (Sigma-Aldrich; Merck KGaA). Aliquots containing 1×105 cells in 100 µl buffer were stained with 10-µl propidium iodide (50 mg/ml) solution and with 5 µl fluorescein isothiocyanate-conjugated Annexin V (17.6 mg/ml; BD Biosciences) for 5 min at 37°C. Following staining, 400 µl PBS was added to the cells. Immediately, flow cytometry analysis was performed using a FACScan flow cytometer (BD Biosciences). All FACS data were analyzed using FlowJo v10 software (FlowJo LLC).
8
Cell Viability and Apoptosis Analysis
9
Apoptosis Assay with Streptochlorin
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Streptochlorin was obtained as previously reported.19 (link) Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum, and other components used for cell culture were purchased from Life Technologies (Grand Island, NY, USA). Fluorescein isothiocyanate-conjugated Annexin V and propidium iodide were purchased from BD Biosciences (Franklin Lakes, NJ, USA). All reagents used were extra-pure grade.
10
Annexin V and PI Apoptosis Assay
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Cells were stained with fluorescein isothiocyanate-conjugated annexin V and propidium iodide (BD, La Jolla, CA, USA) according to the manufacturer’s instructions, and the percentage of apoptotic cells was measured using a FACSCalibur flow cytometer.
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