Vybrant fam poly caspases assay kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Vybrant FAM Poly Caspases Assay Kit is a laboratory tool used to detect and measure the activation of caspases, a group of enzymes involved in programmed cell death. The kit utilizes a fluorescent dye that binds to active caspases, allowing for the quantification of caspase activity in cell samples.

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7 protocols using vybrant fam poly caspases assay kit

Caspase Activation in CAR-T Cells

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The following monoclonal antibodies conjugated with fluorochromes were used: Coxackie-adenovirus receptor, GD2, CD95 (Fas), CD80, CD86, CD40L, OX40L, CD25, CD69, IFN, CD3, granzyme B (BD Biosciences), TRAIL, TRAIL-R1 and TRAIL-R2 (Biolegend). Expression of GD2.CAR by T cells was detected using a specific anti-idiotype, 1A7 (TriGem, Titan), followed by staining with secondary antibody RAM-IgG1 (BD Biosciences). FACS data were collected with a FACSCalibur (Becton Dickinson) and analyzed using FloJo software version 9.3 (Tree Star). For the caspase assay, CHLA-255 cells were seeded in 24-well plates (1 × 10⁵/well), infected with Ad5Δ24 or mock (100 vp/cell), and cultured for 4 days. Control and GD2.CAR-T cells (2.5 × 10⁵/well) were then added to the tumor cells. Active caspases in CHLA-255 cells were measured at 0, 2 and 4 hrs by FACS. The apoptotic cells were stained using Vybrant FAM Poly Caspases Assay Kit (Molecular Probes) according to manufacture’s instructions. The frequency of early apoptotic cells was determined as percentage of FAM⁺ (carboxyfluorescein group as a reporter) cells excluding CD3⁺ and PI⁺ cells from the analysis(32 (link)).

Nishio N., Diaconu I., Liu H., Cerullo V., Caruana I., Hoyos V., Bouchier-Hayes L., Savoldo B, & Dotti G. (2014). Armed Oncolytic Virus Enhances Immune Functions of Chimeric Antigen Receptor-Modified T Cells in Solid Tumors. Cancer research, 74(18), 5195-5205.

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Apoptosis and Cell Cycle Analysis

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Raji or Ramos cells (1 x 10⁶/ml) were incubated with 200 μg/ml FWGP for 1, 3, 6, 12, 24 and 48 hours, washed with PBS and resuspended in 100 μl Annexin-V binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂, pH = 7.4) with 5 μl Annexin-V-Cy5 (BD Pharmingen) and 1 μg/ml Sytox Green (Thermo Fisher) according to the manufacturer’s instructions. After staining for 15 minutes, cells were analyzed by flow cytometry using a FACSCanto instrument (BD); 30,000 events per sample were acquired. Untreated cells were stained as above and used as controls. Untreated, unstained or single-stained controls were used for compensation. To assess caspase activity, cells were incubated with FWGP or PBS control as above and stained with a Vybrant FAM Poly Caspases Assay Kit (Molecular Probes) according to the manufacturer’s instructions. Briefly, 300 μl of cell suspension (1 x 10⁶ cells/ml) were incubated with VAD-FMK FLICA reagent and Hoechst 33342 for the detection of activated caspases 1, 2, 4, 5, 6, 8 and 9, washed and analyzed by flow cytometry as above. Data were analyzed using FlowJo software. For cell cycle analysis, cells were fixed in ethanol, washed, and stained with 20 μg/ml propidium iodide (PI) as previously described [33 (link)]; data (50,000 events/sample) were acquired as noted above.

Barisone G.A., O’Donnell R.T., Ma Y., Abuhay M.W., Lundeberg K., Gowda S, & Tuscano J.M. (2018). A purified, fermented, extract of Triticum aestivum has lymphomacidal activity mediated via natural killer cell activation. PLoS ONE, 13(1), e0190860.

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Analyzing Cell Cycle Progression and Apoptosis in Embryoid Bodies

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For the analysis of cells in the S-phase of the cell cycle, the EBs were incubated with 10 μM 5-ethynyl-2-deoxyuridine (EdU) for 1 h at 37°C and 5% CO₂ and then with a reaction solution from the Click-iT EdU Imaging Kit with Azide-Alexa 488 (C10083; Molecular Probes) according to the protocol recommended by the manufacturer. After the completion of the Click-iT EdU reaction, the EBs were fixed with 3% paraformaldehyde (P6148; Sigma-Aldrich) in PBS, washed with PBS and stained with Hoechst 33342 (1 μg/ml; H3570; Molecular Probes). EBs were cleared in glycerol-PBS, placed on 18-well µ-Slides (81826; Ibidi, Martinsried, Germany) in a mounting medium (50001; Ibidi) and imaged under a Leica TSC SPE confocal microscope.
The Vybrant FAM Poly Caspases Assay Kit (V35117, Molecular Probes) for the detection of caspase-1, -3-9 activity was used according to the manufacturer’s recommendations. The EBs were incubated in culture media containing FLICA reagent (FAM-VAD-FMK poly caspases reagent) for 1 h at 37°C and 5% CO₂ and Hoechst 33342 (1 μg/ml) and propidium iodide (PI; 1 μg/ml) were added to the culture media for 15 min. After washing with PBS, the EBs were mounted on 15-well µ-Slides (81506; Ibidi) and immediately examined under a confocal microscope.

Gordeeva O., Gordeev A, & Erokhov P. (2022). Archetypal Architecture Construction, Patterning, and Scaling Invariance in a 3D Embryoid Body Differentiation Model. Frontiers in Cell and Developmental Biology, 10, 852071.

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Quantifying Caspase-Mediated Apoptosis

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Caspase activities were measured using the Vybrant® FAM Poly Caspases Assay Kit (Molecular Probes). After induction of apoptosis, 150 µL of cell suspensions at 5.10 5 cell.mL - 1 were centrifuged for 8 min at 500 x g at 4°C. Cells were resuspended in FSW. After adding 5 µL of FLICA (Fluorochrome-Labeled Inhibitors of Caspases) 30X, cell suspensions were incubated 1h in the dark at 15°C. Cells were washed twice in 3X washing buffer. Finally, 2 µL of PI were added and suspensions were incubated 10 min on ice in the dark. Four populations were identified depending on the staining: (i) cells that stain positive to FLICA and negative for PI were considered as apoptotic cells (FL1); (ii) cells positive to both FLICA and PI (FL3) were necrotic cells; (iii) cells positive only for PI were considered dead and (iv) cells negative to both FLICA and PI were alive.

Gervais O., Renault T, & Arzul I. (2015). Induction of apoptosis by UV in the flat oyster, Ostrea edulis. Fish & shellfish immunology, 46(2).

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Quantification of Apoptosis in CD34+ Cells

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UCB unit cells were stained for the presence of caspase as per manufacturers instruction (Vybrant FAM Poly Caspases Assay Kit, Life Technologies). Briefly, cells were incubated with FLICA reagent (BD Biosciences) in a FACS tube and then incubated for 60 minutes at 37°C in the dark. The sample was washed twice, stained with CD45APC (BD Biosciences) and CD34PE (BD Biosciences) and incubated for 30 min at 4°C in the dark. After a final wash step, the cells were stained with ViaProbe Cell Viability Solution (7-AAD, BD Biosciences) and the solution was incubated at room temperature. The fractions of viable, early and late apoptotic, and necrotic CD34+ cells were determined. Each tube was spiked with fluorescent beads (PKH, Sigma) to determine the cell losses due to washing. Early apoptotic cells stained positive for caspases but negative for 7AAD. Late apoptotic cells stained positive for both caspases and 7AAD. Necrotic cells were negative for caspases but stain positive for 7AAD and viable cells stain negative for both caspases and 7AAD.

Hubel A., Spindler R., Curtsinger J.M., Lindgren B., Wiederoder S, & McKenna D.H. (2014). Post thaw characterization of umbilical cord blood: markers of storage lesion. Transfusion, 55(5), 1033-1039.

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Caspase Activation in MCF-7 Cells by CGME

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To prove that the cytotoxic effect of CGME mediated caspase activation, we used Carboxyfluorescein poly-caspase (FAM) assay (Life Technologies Inc., USA.).^{21 (link)} The MCF-7 cells treated with CGME (40 μg/ml) for 0, 1, 3, 6, 12 and 24 hours were used for the poly-caspases analysis. The Vybrant® FAM Poly Caspases Assay Kit (Life Technologies Inc., USA.) was used to detect active caspases in apoptotic cells, based on the fluorescent inhibitor of caspases (FLICA™) methodology. The assay was performed with the Attune flow cytometer in accordance with the manufacturer’s instructions.

Kharat KR P.h.D, & Kharat AS P.h.D. (2019). The Calotropis Gigantea Methanolic Extract Induces Apoptosis in Human Breast Carcinoma Cells. Iranian Journal of Medical Sciences, 44(6), 483-492.

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Apoptosis Assay for Frozen PBMCs

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Post thaw apoptosis was determined using PBMCs frozen in TGI cryoprotectant. Cells were thawed rapidly in a water bath for 2 minutes and 30 seconds and then stained for caspases per manufacturer’s instructions (Vybrant FAM poly caspases assay kit, Life Technologies, Carlsbad, CA). Cells were incubated with FLICA reagent for 60 minutes at 37°C and 5% CO₂ in the dark. Cells were washed twice with apoptosis wash buffer, stained for CD4 and CD8, and incubated in the dark at room temperature for 15 minutes. After a final wash step, cells were stained with a viability dye (ViaProbe, 7-AAD, BD Biosciences). Information for each antibody is included in Table 2.
Fractions of viable, early and late apoptotic, and necrotic CD4+ and CD8+ T-cells were determined. Viable cells stained negative for both caspases and 7-AAD. Cells in early apoptosis stained positive for caspases and negative for 7-AAD, while cells in late apoptosis stained positive for caspases and 7-AAD. Necrotic cells stained negative for caspases, but positive for 7-AAD.
The gating strategy identified lymphocytes from forward scattering channel (FSC) and side scattering channel (SSC). A gate was applied to FSC-W and FSC-H to select only single T-cells. CD4+ and CD8+ cells were identified, which indicate helper T-cells and cytotoxic T-cells, respectively. An apoptosis vs 7-AAD plot was created for each cell type.

Pi C.H., Hornberger K., Dosa P, & Hubel A. (2020). Understanding the freezing responses of T-cells and other subsets of human peripheral blood mononuclear cells using DSMO-free cryoprotectants. Cytotherapy, 22(5), 291-300.

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