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Fam yvad fmk

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FAM-YVAD-FMK is a fluorogenic caspase-1 inhibitor. It contains a fluorescent reporter molecule (FAM) and a peptide sequence (YVAD) that targets the active site of caspase-1, a pro-inflammatory cysteine protease. Upon cleavage by caspase-1, the fluorescent reporter is released, allowing for the detection and quantification of caspase-1 activity.

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Lab products found in correlation

Fam devd fmk, by ImmunoChemistry Technologies (3 mentions) Accuri c6, by BD (2 mentions) Facscanto 2, by BD (2 mentions) Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions) Facscalibur, by BD (2 mentions) Poly 1 c, by Merck Group (2 mentions) Synergy 2, by Agilent Technologies (2 mentions) Lsm 510, by Zeiss (2 mentions) Fc500, by Beckman Coulter (1 mentions) Zombie violet, by BioLegend (1 mentions) Caspase glo 1 assay, by Promega (1 mentions) Lps from escherichia coli 0127 b8, by Merck Group (1 mentions) Recombinant human il 1α, by R&D Systems (1 mentions) Propidium iodide, by ImmunoChemistry Technologies (1 mentions) Cytotox 96 kit, by Promega (1 mentions) Axio observer d1 microscope, by Zeiss (1 mentions) Anti il 1β, by R&D Systems (1 mentions) Lsrfortessa flow cytometer, by BD (1 mentions) Lysotracker red, by Thermo Fisher Scientific (1 mentions) 35 mm glass bottom dish, by MatTek (1 mentions)

Close spelling variants of this product

FAM-YVAD-FMK inhibitor probe (3 citations) FAM-YVAD-FMK (FLICA) (3 citations)

34 protocols using fam yvad fmk

1

Caspase-1 Activity Profiling in Sepsis-Associated Bloodstream Infection

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Peripheral blood from patients with SAB and controls (blood donors) was collected in VACUETTE EDTA tubes and stained for caspase‐1 activity with FAM‐YVAD‐FMK (fluorescent‐labeled inhibitor of caspase‐1 [FLICA]; Immunochemistry Technologies, Bloomington, MN) for 1 hr at 37°C, as previously described.8 Leukocytes were labeled with RPE‐CY5‐conjugated mouse antihuman CD45 (DakoCytomation, Glostrup, Denmark), RPE‐conjugated mouse antihuman CD11b (DakoCytomation), and ECD‐conjugated mouse antihuman CD14 (Beckman Coulter; Immunotech, Marseille, France) to differentiate between the various leukocyte populations. Neutrophils and monocytes were separated based on side scatter, CD45, and CD14 gating. Furthermore, the data were confirmed based on side scatter, CD11b, and CD14 gating. Nonspecific binding was analyzed using an isotypic control IgG1 FITC/RPE/RPE‐CY5 (DakoCytomation), and found to be nonsignificant. Caspase‐1 activity was determined via flow cytometry (FC 500 Beckman Coulter, Fullerton, CA) by detecting FLICA fluorescence as mean fluorescence intensity (MFI) value for each sample. Acquisition of data was set to count a total of 50,000 events, and the Kaluza software package was used to analyze the data.
Rasmussen G., Idosa B.A., Bäckman A., Monecke S., Strålin K., Särndahl E, & Söderquist B. (2019). Caspase‐1 inflammasome activity in patients with Staphylococcus aureus bacteremia. Microbiology and Immunology, 63(12), 487-499.
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2

Quantifying Active Caspase-1 and Cell Viability

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Active caspase-1 was quantified by using a FAM-FLICA (fluorescent labeled inhibitor of caspase) detection kit (FAM-YVAD-FMK; ImmunoChemistry Technologies) according to the manufacturer’s instructions. Cell viability was assessed by using Zombie violet (BioLegend). Events were acquired on an imaging flow cytometer (Amnis ImageStream Mark II) and analyzed using IDEAS software.
Lima T.S., Gov L, & Lodoen M.B. (2018). Evasion of Human Neutrophil-Mediated Host Defense during Toxoplasma gondii Infection. mBio, 9(1), e02027-17.
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3

Evaluating Caspase-1 Activation in COVID-19

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To evaluate caspase-1 activation, 5 × 105 PBMCs from COVID-19 patients or healthy donors were centrifuged (400 ×g, 10 min), and cells were labeled for 30 min with the FLICA carboxyfluorescein reagent (FAM–YVAD–FMK; Immunochemistry Technologies), as recommended by the manufacturer. The cells were then washed two times with PBS 1× and fixed with fixative reagent provided by the manufacturer. Acquisition was performed in fixed cells in a flow cytometer (BD Accuri C6; BD Biosciences) and then analyzed using FlowJo (Tree Star) software. To evaluate caspase-1 activity in supernatants, 2 × 105 PBMCs from COVID-19 patients were plated in 96-well plates and incubated overnight. To measure caspase-1 activity, the supernatants were collected, and the Caspase-Glo 1 Assay (Promega) was performed following the manufacturer’s instructions.
Rodrigues T.S., de Sá K.S., Ishimoto A.Y., Becerra A., Oliveira S., Almeida L., Gonçalves A.V., Perucello D.B., Andrade W.A., Castro R., Veras F.P., Toller-Kawahisa J.E., Nascimento D.C., de Lima M.H., Silva C.M., Caetite D.B., Martins R.B., Castro I.A., Pontelli M.C., de Barros F.C., do Amaral N.B., Giannini M.C., Bonjorno L.P., Lopes M.I., Santana R.C., Vilar F.C., Auxiliadora-Martins M., Luppino-Assad R., de Almeida S.C., de Oliveira F.R., Batah S.S., Siyuan L., Benatti M.N., Cunha T.M., Alves-Filho J.C., Cunha F.Q., Cunha L.D., Frantz F.G., Kohlsdorf T., Fabro A.T., Arruda E., de Oliveira R.D., Louzada-Junior P, & Zamboni D.S. (2020). Inflammasomes are activated in response to SARS-CoV-2 infection and are associated with COVID-19 severity in patients. The Journal of Experimental Medicine, 218(3), e20201707.
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4

Inflammasome Activation in RPE and Macrophages

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RPE cells were primed with 4 ng/ml human recombinant IL-1α (R&D Systems, Wiesbaden, Germany) for 48 h [7 (link)] and macrophages with 200 ng/ml lipopolysaccharide (LPS, from Escherichia coli 0127:B8; Sigma-Aldrich, Munich, Germany) for 6 h. For analysis of caspase-1 activity, a fluorochrome-labeled inhibitor of caspase (FLICA) detection assay specific for caspase-1 [carboxyfluorescein-Tyr-Val-Ala-Asp-fluoromethylketone (FAM-YVAD-FMK); Immunochemistry Technologies, Bloomington, MN] was used according to the manufacturer’s instructions. Caspase-1 activation was documented by fluorescence microscopy and quantified by flow cytometry (FACS Canto II; BD Bioscience, Heidelberg, Germany). Interleukin secretion following inflammasome activation in RPE cells was measured by specific ELISAs for human IL-1β (BD Biosciences, Heidelberg, Germany) and human IL-18 (BD Bioscience). Inflammasome activation in murine macrophages was assessed by an ELISA against murine IL-1β (R&D Systems, Wiesbaden, Germany).
Brandstetter C., Mohr L.K., Latz E., Holz F.G, & Krohne T.U. (2015). Light induces NLRP3 inflammasome activation in retinal pigment epithelial cells via lipofuscin-mediated photooxidative damage. Journal of Molecular Medicine (Berlin, Germany), 93(8), 905-916.
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5

Quantifying Cell Death and Cytokine Release

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Cell death was quantified by release of lactate dehydrogenase (CytoTox 96 kit; Promega) and cytokine release by ELISA (R&D Systems or BioLegend). Caspase-1 activation was determined by Fam-YVAD-FMK (ImmunoChemistry Technologies) staining of macrophages infected on glass coverslips, as previously described (35 (link)). Cell permeability was monitored in parallel by staining with propidium iodide (10 μg/ml; ImmunoChemistry Technologies). Caspase-1 activation and cell death were enumerated by counting the fraction of positive cells in at least four separate fields. Subcellular localization of SpeB and IL-1β was examined in macrophages 1 hour after infection with GAS at MOI of 10. Cells were washed in PBS, fixed with Cytoperm (BD Biosciences), incubated with anti-IL-1β (R&D Systems) or anti-SpeB (Toxin Technology, Inc.) antibodies and detected with Alexa Fluor-conjugated secondary antibodies (Invitrogen). Immunofluorescence images were collected using an Axio Observer D1 microscope (Zeiss). Images were processed by identical adjustments for brightness and contrast in ImageJ.
LaRock C.N., Todd J., LaRock D.L., Olson J., O’Donoghue A.J., Robertson A.A., Cooper M.A., Hoffman H.M, & Nizet V. (2016). IL-1β is an innate immune sensor of microbial proteolysis. Science immunology, 1(2), eaah3539.
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6

Detecting Caspase-1 Activation in Monocyte-Derived Dendritic Cells

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Newborn MoDCs were generated as described above and stimulated with individual or combined agonists, as indicated in Figure 3, at 5×105 cells per condition for 6 hours at 37°C. During the last hour of the incubation, a fluorescent Caspase-1 inhibitor, FAM-YVAD-FMK (ImmunoChemistry Technologies, LLC; Bloomington, MN, U.S.A.) was added to detect activated Caspase-1. Cells were subsequently fixed in 4% methanol-free paraformaldehyde and fluorescent intensity at 488 nm was measured by flow cytometry on an LSRFortessa flow cytometer (Beckton Dickinson, San Jose, CA, USA). Data was analyzed with Flowjo software version 10 (Tree Star, Inc., Ashland, OR, USA).
van Haren S.D., Dowling D.J., Foppen W., Christensen D., Andersen P., Reed S.G., Hershberg R.M., Baden L.R, & Levy O. (2016). Age-specific Adjuvant Synergy: Dual TLR7/8 and Mincle Activation of Human Newborn Dendritic Cells Enables Th1-polarization. Journal of immunology (Baltimore, Md. : 1950), 197(11), 4413-4424.
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7

Imaging of Caspase Activation in Infected Cells

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One day before infection (MOI of 10), 1.5 × 105 cells were seeded into a 35-mm glass-bottom dish (MatTek). One hour before imaging, LysoTracker red (100 nM, Invitrogen) was added into the medium. Right before imaging with a spinning-disc confocal microscope, cells were washed with PBS+/+ twice and replaced with Leibovitz’s L-15 medium containing 10% FBS, Oxyrase (1:100), and 10 µg/ml gentamicin. To stain active caspase-1 or caspase-3/7, cells (at 7 hpi) grown on coverslips were incubated with FAM-YVAD-FMK or FAM-DEVD-FMK (Immunochemistry Technologies), respectively. One hour after the incubation, cells were further processed according to the instructions from the manufacturer and mounted on glass slides using ProLong Gold with DAPI.
Yu H.B., Croxen M.A., Marchiando A.M., Ferreira R.B., Cadwell K., Foster L.J, & Finlay B.B. (2014). Autophagy Facilitates Salmonella Replication in HeLa Cells. mBio, 5(2), e00865-14.
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8

Caspase-1 Activation in F. pedrosoi-Infected Macrophages

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After 24 h of infection with F. pedrosoi conidia or hyphae, BMDMs were detached (as mentioned above) and incubated for 1 h with a caspase-1 fluorochrome-labeled inhibitor of caspases (FLICA), FAM-YVAD-FMK (Immunochemistry Technologies), according to the manufacturer’s instructions. Next, cells were washed and stained with APC-conjugated anti-CD11b antibody (eBioscience) in PBS with 2% heat-inactivated FBS (Gibco) to distinguish macrophages from non-internalized fungus. Then, cells were washed and samples were analyzed by flow cytometry as described above.
de Castro R.J., Siqueira I.M., Jerônimo M.S., Basso A.M., Veloso Junior P.H., Magalhães K.G., Leonhardt L.C., de Oliveira S.A., Bürgel P.H., Tavares A.H, & Bocca A.L. (2017). The Major Chromoblastomycosis Etiologic Agent Fonsecaea pedrosoi Activates the NLRP3 Inflammasome. Frontiers in Immunology, 8, 1572.
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9

Assaying Cellular Active Caspase-1 and PI Uptake

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Cellular active caspase-1 content was assayed using the fluorescent inhibitor probe FAM-YVAD-FMK to label active caspase-1 (ImmunoChemistry Technologies) following the manufacturer’s instructions. In brief, FAM-YVAD-FMK was added to cells and incubated for 30 min at 37°C. After extensive washes cell fluorescence was quantified by flow cytometry in FL1 (Gallios; Beckman Coulter). Data were analyzed with the software Kaluza version 1.1.
PI uptake was analyzed by incubating cells with 50 µg/ml PI in PBS in the dark for 5 min. Fluorescence was quantified by flow cytometry in FL3. Data were analyzed with Kaluza version 1.1.
Lordén G., Sanjuán-García I., de Pablo N., Meana C., Alvarez-Miguel I., Pérez-García M.T., Pelegrín P., Balsinde J, & Balboa M.A. (2017). Lipin-2 regulates NLRP3 inflammasome by affecting P2X7 receptor activation. The Journal of Experimental Medicine, 214(2), 511-528.
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10

Cytokine Profiling and Caspase-1 Analysis

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The inflammatory cytokine levels in plasma were investigated using a cytometric bead array mouse inflammation kit (Cat. 552364, BD Biosciences, San Jose, CA, USA), according to the manufacturer's instructions.
For fluorescent labeled inhibitors of CASP assay (FLICA)-CASP1 assay, the flowcytometry (FCM) analysis of adipocytes and SVF cells were conducted according to the previous studies [40 (link),41 (link)]. In brief, epididymal white adipose tissue (eWAT) was minced and digested by type I collagenase (0.8 mg/ml) for 20–30 min. Primary adipocytes and stromal vascular fraction (SVF) cells were separated and stained with FAM-YVAD-FMK (Cat. 98, ImmunoChemistry Technologies, Bloomington, MN, USA) for 2 h, according to the manufacturer's instructions. The stained cells were analyzed using a BD FACS Calibur with Cell QuestPro software (BD Biosciences, San Jose, CA, USA).
Zhang S.Y., Dong Y.Q., Wang P., Zhang X., Yan Y., Sun L., Liu B., Zhang D., Zhang H., Liu H., Kong W., Hu G., Shah Y.M., Gonzalez F.J., Wang X, & Jiang C. (2018). Adipocyte-derived Lysophosphatidylcholine Activates Adipocyte and Adipose Tissue Macrophage Nod-Like Receptor Protein 3 Inflammasomes Mediating Homocysteine-Induced Insulin Resistance. EBioMedicine, 31, 202-216.
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