Cell Viability, Metabolism Assays and Biochemicals

Figure 1. Jurkat cells were treated with DMSO (negative control) or 1 µM staurosporine to induce apoptosis, then stained with TMRE, and photographed using a photomicroscope using a green excitation filter at 510-560 nm and a 570-620 nm emission filter (left). Healthy cells, containing mitochondria with polarized inner membranes fluoresce bright orange (A) compared with apoptotic cells with depolarized mitochondria(B). The corresponding DIC images are shown (right) (Dr. Brian Lee, ICT).

Figure 2. Jurkat cells were exposed to DMSO as the negative control (dark orange bars) or 50 µM CCCP depolarizing agent (light orange bars) and then incubated with TMRE or TMRM. Aliquots (100 µL) were analyzed in triplicate in a black 96-well plate using a fluorescence plate reader (550 nm excitation and 580 nm emission using a 570 nm cut-off filter). Healthy control cells had a high level of orange fluorescence compared to the CCCP-treated cells. (Ms. Tracy Hanson, ICT).

ImmunoChemistry Technologies TMRE - Orange Fluorescent Mitochondrial Membrane Depolarization Assay

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

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.

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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 (Cat. 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. HL-60 cells were treated with camptothecin (Cat. 6210), followed by SR-VAD-FMK (Cat. 916/917) and green FAM-FLISP serine protease inhibitor FFCK (Cat. 946), and then analyzed on a scanning laser cytometer. Active caspases stain red (X-axis) and active serine proteases stain green (Y-axis). Co-localization of caspase and serine protease activity is evident in B, C, D, and G. Data courtesy of Dr. J. Grabarek, Brander Cancer Center, NY, and Pomeranian School of Medicine, Szczecin, Poland.

Figure 2. Paramecium were starved for 12-24 hours in 5 mM KCl to induce autogamy. SR-VAD-FMK (Cat. 916/917), was added to the live Paramecium for 30 minutes in the dark. Cells were then washed and Hoechst 33342 was added. Cells were incubated in the dark for 10 minutes, washed, and applied to coverslips. Images were captured using a Nikon TE-300 Epi-fluorescent microscope with a SPOT-RT digital camera and merged using SPOT software. Data courtesy of Dr. W. E. Bell, Virginia Military Institute.

ImmunoChemistry Technologies Red Fluorescent SR-FLICA® Poly (active) Caspase (VAD) Assay Kit

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Figure 1. Fluorescent Microscopy Analysis of THP-1 Suspension Cells 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.

Figure 2. Caspase Activity in Jurkat Cells Jurkat cells (T lymphocytes) were labeled with our Green Fluorescent Poly (active) Caspase FAM FLICA® Inhibitor Reagent (FAM-VAD-FMK) (Cat. 92) and viewed under a fluorescence microscope. The grey DIC image (B) reveals five cells in the field of view, but only four of them fluoresce green (A). Four out of five cells are apoptotic and have active caspases present (green). Data courtesy of Dr. Brian W. Lee, ICT.

ImmunoChemistry Technologies Green Fluorescent Poly (active) Caspase FAM FLICA® Assay Kit (FAM-VAD-FMK)

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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).

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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

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Figure 2. Phosphate standard curve was generated using fluorometric detection: excitation 535 nm and emission 585 nm. Incubation time = 60 minutes at 37°C. Standard curve range 1.5625 μM to 100μM. Graph was plotted using 4PL non-linear regression.

ImmunoChemistry Technologies Fluorescent Phosphate Detection Kit

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ImmunoChemistry Technologies Fluorescent Monoamine Oxidase Detection Kit

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ImmunoChemistry Technologies Fluorescent Lactate Detection Kit

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Figure 1. Staurosporine induced Jurkat cell viability assay. Titrated doses of staurosporine were added into seeded Jurkat cells (10,000 cells/well) in 96-well black opaque tissue culture plates and incubated at 37°C, 10% CO2 for 24h. Dye reagent (1/10 volume) was added into the cultured cells and incubated for 3h and fluorescence was detected at Ex: 530 nm and Em: 590 nm.

Figure 2. PBMC proliferation in response to mitogen Concanavalin A (Con-A) stimulation measured using Fluorescent Cell Proliferation Assay. PBMC at 15,000 cells were cultured for 3 days in the presence of titrated amounts of Con A and tested for proliferation using the Fluorescent Cell Proliferation Assay. Dye reagent (1/10 volume) was added to the cultured cells and incubated at 37°C for 2h. Fluorescence was detected at Ex: 530 nm and Em: 590 nm.

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Figure 2. The reaction contained 20mM hydrogen peroxide (final) per well and the indicated amounts of catalase. The reaction was incubated for 30 minutes at room temperature. Reaction cocktail was added to each well and the reaction incubated for another 10 minutes in the dark at room temperature. Fluorescence was measured at excitation 530nm and emission 590nm. The graphs show the net fluorescence change: Negative Control fluorescence (no catalase) minus Sample (Cat.alase) fluorescence.

ImmunoChemistry Technologies Fluoro Catalase Activity Detection Kit by Cell Technology

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ImmunoChemistry Technologies Fluorescent ATP Detection Kit

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ImmunoChemistry Technologies Fluorescent ADP Detection Kit

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ImmunoChemistry Technologies Fluoro AChE - a fluorescence assay for monitoring and detecting acetylcholinesterase activity in samples

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Figure 1. Standard curve (fluorescence). Excitation 535 nm and emission 585 nm. R2 value=0.9977. Incubation time= 1 hour at room temperature. Standard curve range 0.15625 μM to 10μM. A new curve must be generated each time the assay is run.

Figure 2. Standard curve (absorbance). Absorbance read-out at 570 nm. R2 =0.9993. Incubation time = 30 minutes. Standard curve range 1.25μM to 80μM. A new curve must be generated each time the assay is run.

ImmunoChemistry Technologies Fluorescent Total Cholesterol Detection Kit

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Figure 2. Comparison of NADPH & NADH Standard Curves

ImmunoChemistry Technologies Fluorescent NADP/NADPH Detection Kit

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ImmunoChemistry Technologies Chlorination and Oxidation detection Kit

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Figure 1. Jurkat cells were stimulated with Staurosporine for 3 hours (B) or DMSO (A). Mito Flow reagent was added according to the protocol, incubated for 30 minutes and analyzed by flow cytometry: Ex:488nm Em: FL2 channel.

ImmunoChemistry Technologies Mito Flow - Mitochondrial Membrane Potential Detection Kit

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Figure 2. Detection of Hypochlorite ( -OCl ) in neutrophils. Neutrophils were isolated from porcine blood, washed in Krebs-Ringers phosphate buffer and seeded in glass bottom dishes. The cells were then loaded with APF (10μM final) by incubation for 30 minutes at room temperature. The Dye-loaded neutrophils were stimulated with PMA (2ng/mL). Fluorescence images were acquired before and 10 minutes after stimulation. Excitation: 488nm emission: 505-550 nm barrier filters

ImmunoChemistry Technologies Highly Reactive Oxygen Species (hROS) Detection Kit

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Figure 1. Detection of Hypochlorite ( -OCl ) in neutrophils. Neutrophils were isolated from porcine blood, washed in Krebs-Ringers phosphate buffer (as described in step V 1. above) and seeded in glass bottom dishes. The cells were then loaded with APF or HPF (10 mM final) by incubation for 30 minutes at room temperature. The Dye-loaded neutrophils were stimulated with PMA (2ng/mL).

Figure 2. Fluorescence images were acquired before and 10 minutes after stimulation. Excitation: 488 nm emission: 505-550 nm barrier filters. Hypochlorite production can be detected with increase APF fluorescence and no increase in HPF fluorescence.

ImmunoChemistry Technologies Fluorescent Hypochlorite Detection Kit

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Figure 1. Jurkat cells were stimulated with staurosporine (B) or DMSO (A), then stained according to the kit protocol. The cells were washed twice and analyzed by flow cytometry: Ex:488nm Em: FL1 and FL2. A. Healthy cells show a strong red fluorescence (intact mitochondria) and no green fluorescence (no active caspases). B. Apoptotic cells show a loss of red fluorescence (y axis) due to loss of mitochondrial membrane potential and positive green fluorescence (x axis) indicating active caspases.

Figure 2. HeLa Cells activated with staurosporine (B) or DMSO (A), then stained according to the kit protocol. The cells were washed twice and analyzed by flow cytometry: Ex:488nm Em: FL1 and FL2. A. Healthy cells show a strong red fluorescence (intact mitochondria) and no green fluorescence (no active caspases). B. Cells becoming apoptotic show loss of red fluorescence (y axis) due to loss of mitochondrial membrane potential and positive green fluorescence (x axis) indicating active caspases.

ImmunoChemistry Technologies Dual Caspase 1 and Mitochondrial Membrane Potential Assay Kit

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ImmunoChemistry Technologies Dual Caspase 3/7 and Mitochondrial Membrane Potential Assay Kit

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ImmunoChemistry Technologies Dual Poly Caspase and Mitochondrial Membrane Potential Assay Kit

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Cell Viability, Metabolism Assays and Biochemicals

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