A FAM-FLICA Poly Caspase Assay Kit (ImmunoChemistry Technologies, LLC, Bloomington, MN, USA) was used to measure the level of caspase activation in oocytes according to the manufacturer's protocols. In brief, oocytes were incubated with the reaction mix in a humidified atmosphere of 5% CO2 at 39°C for 30 min. After incubation, oocytes were washed three times in PBS-PVA.
Fam flica poly caspase assay kit
Manufactured by ImmunoChemistry Technologies
Sourced in United States
The FAM-FLICA™ Poly Caspase Assay Kit is a laboratory tool that detects and measures the activation of multiple caspases, which are enzymes involved in programmed cell death. The kit uses a fluorescent-labeled inhibitor of caspases (FLICA) to label activated caspases in cells, allowing for their quantification and analysis.
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Lab products found in correlation
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (3 mentions)
Facscalibur, by BD (2 mentions)
Facscanto 2, by BD (2 mentions)
Fixable viability dye efluor 506, by Thermo Fisher Scientific (2 mentions)
Annexin 5 apoptosis detection kit, by Thermo Fisher Scientific (2 mentions)
Eflour450, by Thermo Fisher Scientific (2 mentions)
Ki 67 kit, by BD (2 mentions)
Propidium iodide, by Merck Group (1 mentions)
Lsrfortessa, by BD (1 mentions)
Hoechst 33342, by ImmunoChemistry Technologies (1 mentions)
Fam flica caspase 1 assay kit, by ImmunoChemistry Technologies (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Facsaria 3, by BD (1 mentions)
Perm wash, by BD (1 mentions)
Mitotracker green, by Thermo Fisher Scientific (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Mtt assay kit, by American Type Culture Collection (1 mentions)
Losartan, by Merck Group (1 mentions)
11 protocols using fam flica poly caspase assay kit
1
Caspase Activity Assay in Oocytes
2
Cell Cycle Analysis and Apoptosis Detection
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For cell cycle analysis, cells were washed twice with PBS and fixed with cold 70% ethanol. After incubation with 10 μg/ml RNase and 50 μg/ml propidium iodide (PI) (Sigma-Aldrich) at 37 °C for 30 minutes. For detection of apoptotic cells 1 × 106 cells were double stained with CF633-conjugated annexin V (Biotium) and PI for 15 min at room temperature. Binding buffer was added before analysis. For detection of caspase activity, cells were stained using FAM FLICA poly caspase assay kit (Immunochemistry technologies) as per manufacturer instructions. Data acquisition was obtained using BD LSR Fortessa (BD Biosciences) flow cytometer. Fluorescence signals were collected at the wavelengths of 610/20 nm for PI, 670/14 nm for annexin V, and 530/30 nm for FLICA. The data was analyzed using the FACSDiva software.
3
Detecting Apoptotic Cells by Caspase Assay
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Apoptotic cells were detected using the FAM-FLICA Poly Caspase Assay Kit (ImmunoChemistry Technologies), and cells were counterstained with Hoechst 33342 (200 µg/ml, ImmunoChemistry Technologies).
4
Dendritic Cell Phenotype and Apoptosis
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DCs were stimulated as described above and at 24 hours, cells were harvested and stained with fluorescently-labeled antibodies to CD80, CD86 and MHC class II, (all from BD Biosciences), or Annexin V-FITC and propidium iodide (PI) using the Annexin V-FITC Apoptosis Detection Kit according to the manufacturer’s protocol (BD Pharmingen). Early apoptotic cells were defined as Annexin V+ and PI-, and necrotic cells were defined as Annexin V+ and PI+. For the measurement of caspase activity, the FAM-FLICA™ Poly Caspase Assay Kit was used and for the measurement of active caspase-1, the FAM-FLICA™ Caspase 1 Assay Kit (both from ImmunoChemistry Technologies) was used. Cells were analyzed by flow cytometry (Gallios™ flow cytometer, Beckman Coulter) and at least 5000 cells were counted.
5
Multiparametric Analysis of Lymphocyte Apoptosis
6
Fc Receptor Blocking and Apoptosis Assay
7
Caspase Activation by PpIX Assay
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Activation of caspases by PpIX was measured with FAM-FLICA™ Poly Caspase Assay Kit (ImmunoChemistry Technologies, Germany) according to manufacturer’s protocol. Caspase activation was monitored by the means of flow cytometry with BD FacsCalibur after 6 h treatment with PpIX.
8
Quantifying Apoptosis and Necrosis in Cortical Neurons
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To evaluate caspase activity in cortical neurons after OGD, cells were incubated in fluorochrome-labeled inhibitor of caspases (FLICA) solution (Immunochemistry Technologies, FAM-FLICA® Poly Caspase Assay Kit) at 37°C for 1 h. After three washes, cells were labeled with propidium iodide (PI) and immediately imaged using a fluorescence microscope. Cell nuclei were counter-stained with Hoechst 33342. PI labeling was used with FLICA to identify four populations of cells: living (FLICA–, PI–); early apoptotic (FLICA+, PI–); late apoptotic (FLICA+, PI+); and necrotic (FLICA–, PI+). Unstained live cells were labeled with only Hoechst 33342, and necrotic cell membranes appeared compromised and stained with PI in red. Cells in the late apoptotic phase were dually stained with FAM-FLICA (green) and PI, and cells in the early apoptotic stage were stained only with FAM-FLICA. Apoptotic/necrotic cells were counted using a microscope (Nikon Eclipse Ti, Nikon Instruments Inc., Japan), and the proportion of apoptotic/necrotic cells /total number of cells ×100% was calculated (containing total number of cells ranging from 321 to 556 cells).
9
Multiparametric Flow Cytometry Analysis
10
Multiparametric Flow Cytometry Analysis
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Supernatant of hybridoma 2.4g2 (anti-CD16/32) was used to block Fc receptor. Cells were stained with Fixable Viability Dye eFluor 506 (eBioscience) to detect dead cells before surface staining. Annexin V apoptosis detection kit, eFlour 450 (eBioscience) and Ki-67 kit (BD Biosciences) were used according to manufacturers’ protocol. Antibodies used for flow cytometry were listed in Supplementary Table 1 . Pan-caspase activity was detected by FAM-FLICA™ Poly Caspase Assay Kit (ImmunoChemistry Technologies) according to manufacturer's protocol. For intracellular cytokine staining and transcription factor staining, Foxp3/Transcription Factor Staining Buffer Set (eBioscience) was used as manufacturer’s protocol. IELs (1 × 106 cells/ml) and LPLs (1 × 106 cells/ml) were stimulated with IL-12 (10 ng/mL) or IL-23 (10ng/mL)/IL-1β (10ng/mL) with GolgiPlug at 37°C for 4 hours for cytokine production, or IL-15 (20 ng/mL) at 37°C for 24 hours for Bcl-2 expression. Data were acquired on a FACSCanto II (BD Biosciences) and analyzed with FlowJo software (TreeStar).
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