Cell Viability, Metabolism Assays and Biochemicals

Figure 1. THP-1 cells were treated with either a negative control (A), or PMA (5 ng/mL) followed by LPS (10 ng/mL). Cells were then stained with FAM-YVAD-FMK (cat. 97/98) and analyzed by phase contrast and fluorescence microscopy. In the treated sample, many cells appear bright green, indicating increased caspase-1 activity (B). In the non-induced sample, few green cells are visible, indicating a low level of caspase-1 activity (A). Data courtesy of Dr. Brian Lee, ICT (207:11).

RAW264.7 monocytes were exposed to varying concentrations of CdCl2 (0 μM, 10 μM,15 μM) for 24 h, as well as a positive control group (10 μM nigericin). Cells were stained with FAM-FLICA (Cat no 98). Images of cells were taken under brightfield and, after exposure, to fluorescence (400× magnification). Image from publication. CC-BY-4.0 PMID: 39408668

ImmunoChemistry Technologies Green Fluorescent FAM-FLICA® Caspase-1 (YVAD) Assay Kit

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Figure 1. Cresyl Violet Perchlorate Excitation and Emission Spectra in Ethanol

Figure 2. MCF-7 cells were stained with acridine orange for 30 minutes, then washed twice in PBS (cells not stained with Magic Red®) and then analyzed by fluorescence microscopy at 400X using either 492 nm excitation with a 540-550 nm emission filter (A, lysosomes appear yellowish green), or 540 nm excitation with a long pass >640 nm barrier filter (B, lysosomes appear red). Data from the laboratory of Dr. Z. Darzynkiewicz (Brander Cancer Research Center Institute, New York City, NY).

ImmunoChemistry Technologies Magic Red® Fluorescent Cathepsin B Assay Kit

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Figure 1. Signals leading to activation of the NLRP3 inflammasome.

Figure 2. C2BBe1 cells were infected with wild type Salmonella constitutively expressing mCherry (red). After 9 hours, live cells were incubated with FAM-YVAD-FMK (green cell) for 1 hour. The infected cell (red) is undergoing pyroptosis, and shows increased green fluorescence compared to uninfected cells due to caspase-1 activation. Data courtesy Knodler, Leigh A., et al. Dissemination of invasive Salmonella via bacterial-induced extrusion of mucosal epithelia. PNAS. 107:41, 17733-17738 (2010).

ImmunoChemistry Technologies Pyroptosis/Caspase-1 Assay - Green Fluorescent

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Figure 1. FLICA® 660 was reconstituted in DMSO, diluted in diH2O, and analyzed in a fluorescence plate reader. The excitation spectrum was generated using an emission of 760 nm. The emission spectrum was generated using an excitation of 600 nm.

Figure 2. Flow cytometry analysis of Jurkat cells treated with FLICA® 660-YVAD-FMK. After spiking FLICA® into the cell culture media, the cells were treated with either a negative control (left) or nigericin (20 μM, no.6698) to induce caspase-1 activity (right) for 24 hours. The negative control induced caspase-1 activity in 6.8% of cells (left); treatment with nigericin induced caspase-1 activity in 57.3% of cells (right). This is a ratio of 8:1. Data courtesy of Mrs. Tracy Murphy, ICT, 230-01.

ImmunoChemistry Technologies Far-Red Fluorescent FLICA® 660 Caspase-1 (YVAD) Assay Kit

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Figure 1. Flow cytometry analysis of active caspase-9 in MDA-MB-231 (A,B) and ZR-75-1 (C,D) cells. Cells were stained with FAM-FLICA® Caspase-9 reagent. From Figure 5 of R. Krętowski and M. Cechowska-Pasko, The Reduced Graphene Oxide (rGO) Induces Apoptosis, Autophagy and Cell Cycle Arrest in Breast Cancer Cells, Int J Mol Sci (2022) 23, 9285. Used under the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). Copyright © 2022 by the authors.

Flow cytometry analysis of the caspase-9 activity (Assay Kit, Cat no. 912) in the MCF-7 (A,B) and MDA-MB-231 (C,D) breast cancer cells after 24 h of incubation with Les-6287 (1.0 μM and 1.5 μM), doxorubicin (1.0 μM), and DMSO. Image from publication, CC-BY-4.0, PMID: 39199694.

ImmunoChemistry Technologies Green Fluorescent FAM-FLICA® Caspase-9 (LEHD) Assay Kit

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Figure 1. MR-(DEVD)2 (cat. 936) and Hoechst 33342 were used to quantify apoptosis in human PASMCs. Cells were treated with a negative control (A), a drug that inhibits proliferation (B), or H2O2 to induce apoptosis (C). Apoptotic cells stained red with cleaved MR-(DEVD)2 and blue with Hoechst. They have less intense blue nuclei than healthy cells, which have bright blue nuclei. Research of Dr. F. Perros, et al., Universite Paris-Sud 11; Clamart; Hopital Henri-Mondor, Creteil, France.

Figure 2. THP-1 cells were stained with Poly (active) Caspase FAM FLICA® Inhibitor Reagent (green), and Hoechst 33342 (Blue). Cells were incubated with staurosporine to induce apoptosis. Two photos were taken and superimposed. Only one cell of the three cells is apoptotic (middle) – it is stained positive for caspase activity with FAM-VAD-FMK. Data courtesy of Dr. Brian W. Lee.

ImmunoChemistry Technologies Hoechst 33342 Fluorescent Nucleic Acid Stain

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Figure 1. Using FAM-FLICA®, non-apoptotic cells (unstained, left of histogram) can easily be distinguished from apoptotic cells exhibiting green fluorescence (right of histogram). Jurkat cells were either treated with DMSO as control (A), or 1 µM staurosporine (B) to induce apoptosis. Cells were stained with FAM-FLICA® and analyzed using a flow cytometer in FL-1. Only 7% of non-induced cells (A, right) are apoptotic compared with 96.5% of the induced cells (B, right). (ICT 226:17-19.)

Figure 2. Using 7-AAD, live cells (unstained, left side of each histogram) can easily be distinguished from dead/membrane compromised cells exhibiting red fluorescence (right side of each histogram). Jurkat cells were either untreated (A), or treated with 90% ethanol for 60 seconds, then stained with 7-AAD, and analyzed using a flow cytometer in FL-3. Only 4% of untreated cells (A) are dead compared with 97.6% of the treated cells (B). Data courtesy of Dr. Kristi Strandberg, ICT 226:30-31.

ImmunoChemistry Technologies Necrosis vs Apoptosis Assay Kit

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ImmunoChemistry Technologies Cell-mediated Cytotoxicity Assay: Basic Cytotoxicity Test

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Figure 1. Monocytes can secrete active caspase-1, therefore cells were labeled with FAM-FLICA® Caspase-1 (WEHD) (cat. 9161/9162) prior to treatment. Cells were then treated with DMSO (control) or 10 µM nigericin (cat. 6698), stained with Hoechst 33342, fixed (cat. 636), and viewed using EGFP (Ex 470/30, Em 530/50) and DAPI (Ex 375/28, Em 460/50) LED filter cubes at 20X. Increased levels of FAM-FLICA® staining were seen in the nigericin-treated cells. Data courtesy of Dr. Kristi Strandberg, ICT (235:60).

ImmunoChemistry Technologies Green Fluorescent FAM FLICA® Caspase-1 (WEHD) Assay Kit

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Figure 1. Suspension Jurtkat cells were treated with DMSO (control, A) or 1 µM staurosporine (B) for 4 hours at 37°C, then stained with SR-LETD-FMK (kit cat. 9149/9150) for 1 hour at 37°C. Cells were washed three times and slides prepared. Treated cells appear bright red, indicating a high level of caspase-8 activity (B). Few non-induced cells are red, indicating minimal caspase-8 activity (A, Non-Induced, left). Data courtesy of Mrs. Tracy Murphy, ICT (220:68, 121815).

ImmunoChemistry Technologies Red Fluorescent SR-FLICA® Caspase-8 (LETD) Assay Kit

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ImmunoChemistry Technologies Magic Red® Fluorescent Cathepsin L Assay Kit

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Figure 2. MCF-7 cells were exposed to 0.15 µM camptothecin (cat. 6210) for 24 hours at 37°C, then stained with MR-(DEVD)2 for 30 minutes at 37°C, washed, and then stained with 1 µg/mL Hoechst stain. Apoptotic cells with orange-red lysosomal bodies and less intense blue nuclei are intermixed with non-apoptotic cells bearing bright blue nuclei and absent or reduced orange-red lysosomal staining. Photo provided by Dr. Zbigniew Darzynkiewicz at Brander Cancer Research Center Institute, New York, NY.

ImmunoChemistry Technologies Magic Red® Fluorescent Caspase-3/7 Assay Kit

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Figure 1. Cell death in primary rat hippocampal neurons. (A) is a composite of FLICA® FAM-DEVD-FMK (B), PI (C), and Hoechst (D). DNA is labeled in 4 cells (D), 3 cells are caspase-positive (B), 2 are membrane-compromised (C). One cell is in early apoptosis (green but not red). Two FLICA®-positive cells are also PI-positive (red, C) (becoming membrane compromised) and are in the late stages of apoptosis rather than necrosis. Data courtesy of Dr. Z. Kahraman Akozer, University of Maryland.

Figure 2. Normal (A) and keratoconus (B) corneal fibroblasts were treated with 200 μM H2O2 for 1 hour, washed, and allowed to recover. Cells were then treated with FAM-DEVD-FMK (cat. 93/94) for 1 hour, then washed with 1X Apoptosis Wash Buffer. Keratoconus corneal fibroblasts treated with H2O2 (B) show a significant increase in caspase-3/7 activity compared to normal cells (A). Non-apoptotic cells are dark in background. Data courtesy of Dr. Cristina Kenney, University of California, Irvine.

ImmunoChemistry Technologies Green Fluorescent FAM-FLICA® Caspase-3/7 (DEVD) Assay Kit

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Figure 1. Assay principle. The kit uses a non-fluorescent detection reagent, which is reduced by NADH to produce its fluorescent analog and NAD. NAD is further converted to NADH via an enzyme coupled reaction (which does not react with NADP/NADPH).

Figure 2. NADH standard curve. Titrated in NADH standard diluent. Incubation time was 1 hour.

ImmunoChemistry Technologies Fluoro NAD/NADH Detection Kit by Cell Technology

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Figure 1. (a) Fluorescent responses of NP3 (5 μM) toward various analytes (10 μM). Data shown represent fluorescent intensity at 470nm, 30 min after addition of the analytes. (b) ONOO− (final 10 μM) was quickly injected into a solution of NP3 (final 5μM), and the fluorescent intensity at 470 nm was plotted against time. (c) Fluorescence enhancement of NP3 (5μM) at 470 nm as a function of ONOO− (0−10 μM) after 15 min of reaction. All data acquired in PBS (10 mM, pH 7.4) with excitation at 375 nm.

Figure 2. (a) Time-lapse images taken from living EA.hy926 endothelial cells. Cells were preincubated with NP3 (5.0 μM), followed by stimulation with or without SIN-1 (0.5 mM). (b) Dynamic changes of NP3 fluorescence after SIN-1 (0.5 mM) treatment in panel a. (c) Effects of ONOO− scavengers uric acid (100 μM) and FeTTPS (1 μM) on changes in NP3 fluorescence in endothelial cells in the presence of ONOO− (60 μM). PI (red) stains nuclei. NP3 fluorescence was collected at 420−480 nm with λex 405 nm.

ImmunoChemistry Technologies Peroxynitrite Detection Kit

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Figure 1. Assay principle. The kit uses a non-fluorescent detection reagent to measure H2O2. H2O2 oxidizes the detection reagent in a 1:1 stoichiometry to produce a fluorescent product resorufin.

Figure 2. Typical Hydrogen peroxide standard curve. A new curve must be generated each time the assay is run.

ImmunoChemistry Technologies Hydrogen Peroxide Detection/Peroxidase Detection Kit

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Figure shows the two phosphate moieties of ADP, with no trace of a peak indicating the third phosphate of ATP using P31 NMR.

ImmunoChemistry Technologies Ultra Pure ADP by Cell Technology

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Figure 1. Propidium iodide excitation and emission spectra.

Figure 2. HL-60 cells were treated with a drug, then stained with FLICA® FAM-VAD-FMK poly-caspase inhibitor reagent (green) (Cat. 92) and propidium iodide (red) (PI). Scanning laser cytometer analysis. Four populations of cells were detected: (A) Unstained live cells, (B) red necrotic cells (PI), (C) green and red late apoptotic cells (FAM-FLICA® plus PI), and (D) green early apoptotic cells (FAM-FLICA®). Data courtesy of Dr. Z. Darzynkiewicz, Brander Cancer Center, NY.

ImmunoChemistry Technologies Propidium Iodide Stain

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Figure 2. Jurkat cells were treated with a negative control (left) or staurosporine, an apoptosis-inducing agent (right), for 4 hours, then stained with FLICA® 660-DEVD-FMK (cat. 9125) for 1 hour. Cells were washed and analyzed by flow cytometer. The negative control induced caspase-3/7 activity in 4.4% of cells (left); treatment with staurosporine induced caspase-3/7 activity in 70.9% of cells (right). This is a ratio of 16:1. Data courtesy of Mrs. Tracy Murphy, ICT, 13F38, 022013.

ImmunoChemistry Technologies Far-Red Fluorescent FLICA® 660 Caspase-3/7 (DEVD) Assay Kit

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Figure 2. Intracellular cathepsin activity was detected in HL60 cells using Magic Red®-(LR)2 cathepsin K fluorogenic substrate. Intracellular localization of the hydrolyzed fluorescent Magic Red® product was detected using a Nikon Eclipse E800 photomicroscope equipped with a 510–560 nm excitation filter and a 570–620 nm emission/barrier filter at 500X (A). Photo at right (B) shows the corresponding DIC image of the cells. Data courtesy of Dr. Brian Lee, ICT, 061202.

ImmunoChemistry Technologies Magic Red® Fluorescent Cathepsin K Assay Kit

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Figure 1. Control Alzheimer's Disease (AD) macrophages and curcumin-treated AD macrophages were exposed to FITC-labeled Aß and stained with SR-DEVD-FMK (#931/932). Macrophages that engulf FITC-Aß fluoresce green (right). Caspase-positive cells fluoresce red (left). Essentially all of the untreated AD macrophages engulfed FITC-Aß and became apoptotic. Curcumin-treated AD macrophages engulfed FITC-Aß but did not become apoptotic. Data courtesy of Dr. Milan Fiala, UCLA 072205.

ImmunoChemistry Technologies Red Fluorescent SR-FLICA® Caspase-3/7 (DEVD) Assay Kit

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Figure 1. Assay principle: a non‐fluorescent detection reagent is oxidized in the presence of hydrogen peroxide and MPO to produce its fluorescent analog.

Figure 2. A MPO standard curve was prepared and run, as described in the protocol, in the absence (A) or presence (B) of 20 mM catalase inhibitor. MPO standard and reaction mix were added to a 96-well black plate and incubated at room temperature in the dark for 30 minutes. The wells were read using Ex: 530nm and Em: 590nm. There is an approximately 50% reduction in signal in the presence of 20mM catalase inhibitor (note the different scales on the x-axis).

ImmunoChemistry Technologies Myeloperoxidase Detection Kit

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Figure 2. Typical standard curve. A new curve must be generated each time the assay is run.

ImmunoChemistry Technologies Fluorescent Sarcosine Detection Kit

$368

Figure 2. Bovine serum Semicarbazide-sensitive amine oxidase was serially diluted in 1X Reaction buffer. The serially diluted samples were run as described in the protocol. The samples were read after a 3 hours incubation period. Excitation: 530 nm and emission: 590 nm.

ImmunoChemistry Technologies Fluorescent Semicarbazide-Sensitive Amine Oxidase Detection Kit

$490

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Cell Viability, Metabolism Assays and Biochemicals

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