Substrates for Microsomal Dealkylases, Acetyltransferases, Luciferases and Other Enzymes - Section 10.6

Metabolic oxidation of chemical compounds, including many pollutants, is the function of the cytochrome-mediated monooxygenase or mixed-function oxidase system. Several enzymes are involved, including cytochrome P448 monooxygenase (aryl hydrocarbon hydroxylase), which is induced by carcinogenic polyaromatic hydrocarbons. Cytochrome P450 is a useful marker of endoplasmic reticulum membranes. The very low turnover rate of these enzymes can be followed using various fluorogenic alkyl ether derivatives of coumarin, resorufin and fluorescein, all of which yield cleavage products with longer-wavelength spectral properties than the parent substrates.

Resorufin-Based Microsomal Dealkylase Substrates

The four resorufin ether–based substrates (R351, R352, R441, R1147), which all yield fluorescent resorufin (R363, Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1; excitation/emission maxima ~571/585 nm, ), have been extensively used to differentiate isozymes of cytochrome P450. Ethoxyresorufin O-deethylase and total protein concentration have been simultaneously assayed in a fluorescence microplate reader using resorufin ethyl ether (ethoxyresorufin, R352) and fluorescamine (F2332, F20261; Quantitation and Selective Purification of Proteins in Solution—Section 9.2).

Coumarin-Based Microsomal Dealkylase Substrates

Fluorescence detection of the deethylation of 3-cyano-7-ethoxycoumarin (C684) is reported to be 50–100 times more sensitive than that of ethoxyresorufin, primarily because of the faster turnover rate of 3-cyano-7-ethoxycoumarin; however, ethoxyresorufin exhibits lower fluorescence background due to its more favorable spectral shifts. The deethylase product of 3-cyano-7-ethoxycoumarin, 3-cyano-7-hydroxycoumarin (C183, Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1), has a lower pK_a than that of 7-ethoxycoumarin, allowing continuous measurements of enzyme activity at pH 7.

The cytochrome P450 substrate 7-ethoxy-4-trifluoromethylcoumarin (E2882) yields a product with a fluorescence emission that is distinct from that of the substrate and of NADPH, making this substrate useful for the direct measurement of enzymatic activity. Researchers have shown that this substrate is cleaved by at least the 1A2, 2E1 and 2B1 isozymes of cytochrome P450.

Other Microsomal Dealkylase Substrates

The fluorescent products of most microsomal dealkylase substrates rapidly leak from live cells, making them ineffective for measuring intracellular enzymatic activity by imaging or flow cytometric analysis. Using techniques for product retention that proved successful for our Patented DetectaGene Green, ImaGene Green and ImaGene Red glycosidase substrates, we have developed unique substrates that can potentially be used to detect dealkylase activity in single cells.

Like the DetectaGene CMFDG β-galactosidase substrate (D2920, Detecting Glycosidases—Section 10.2), the mildly thiol-reactive chloromethyl moiety of 5-chloromethylfluorescein diethyl ether (C6533) reacts with glutathione or other intracellular thiols to produce a product that is retained in cells through cell division. Adding a chloromethyl moiety to our glycosidase and peptidase substrates (Detecting Glycosidases—Section 10.2, Detecting Peptidases and Proteases—Section 10.4; Figure 10.17) has enabled researchers to identify cells with enzymatic activity 24 hours after loading the substrate. Fluorescein ethers are known microsomal dealkylase substrates. Chloromethylfluorescein diethyl ether was used to monitor cytochrome P450–dependent mixed function oxidase activity in cultured hepatocytes.

Figure 10.17 Sequential β-galactosidase hydrolysis and peptide conjugate formation of CMFDG, a component of the DetectaGene Green CMFDG lacZ Gene Expression Kit (D2920).

Lipases play an essential role in the transfer of lipids in cell signaling and metabolism and generally include glycerol ester hydrolases and cholesterol esterases. Phospholipase A selectively hydrolyzes lipophilic esters of phospholipids. Because of their importance to the process of signal transduction in cells, our extensive selection of substrates and other probes for phospholipases is discussed in Probes for Lipid Metabolism and Signaling - Section 17.4.

EnzChek Lipase Substrate

The triacylglycerol-based EnzChek lipase substrate (E33955) offers higher throughput and better sensitivity than chromogenic (TLC or HPLC) assays, and a visible wavelength–detection alternative to pyrene-based fluorescent substrates. In the presence of lipases, the nonfluorescent EnzChek lipase substrate produces a bright, green-fluorescent product (excitation/emission maxima of ~505/515 nm) for the accurate and sensitive detection of lipase activity in solution. Furthermore, the green-fluorescent product of the EnzChek lipase substrate exhibits pH-insensitive spectra in the physiological pH range and is compatible with optics used for fluorescein detection in fluorometers.

Coumarin-Based Lipase Substrates

The fluorogenic lipase substrate O-pivaloyloxymethyl umbelliferone (C-POM, P35901) was developed to deliver optimal performance in assays of lipase activity. Standard lipase substrates may exhibit high levels of undesirable nonspecific reactivity, either through spontaneous hydrolysis or direct reaction of the substrate with noncatalytic proteins such as BSA. C-POM is much less prone to these unwanted side reactions, and the resulting low level of background fluorescence yields a better signal-to-noise ratio, providing a more accurate measure of lipase catalysis. Enzymatic conversion of the essentially nonfluorescent C-POM yields a bright blue-fluorescent reaction product (excitation/emission ~360/460 nm). C-POM has been shown to serve as a substrate for a variety of lipases and displays excellent stability in solution, making it an ideal substrate for specific lipases or for general or high-throughput screening.

Unlike lipase substrates that are esters of 7-hydroxy-4-methylcoumarin (β-methylumbelliferone, H189; Introduction to Enzyme Substrates and Their Reference Standards - Section 10.1) — a dye that is not appreciably fluorescent at neutral pH — 6,8-difluoro-4-methylumbelliferyl octanoate (DiFMU octanoate, D12200) can be used for the continuous in vitro assay of lipases at a pH greater than or equal to 6; the blue-fluorescent hydrolysis product of DiFMU octanoate, 6,8-difluoro-7-hydroxy-4-methylcoumarin (DiFMU, D6566; Introduction to Enzyme Substrates and Their Reference Standards - Section 10.1; ), has a pK_a of 4.9.

Cholesterol Esterase Assay

The cholesterol produced by cholesterol esterases is readily quantitated using the Amplex Red Cholesterol Assay Kit (A12216), which is discussed in Substrates for Oxidases, Including Amplex Red Kits - Section 10.5 and Sphingolipids, Steroids, Lipopolysaccharides and Related Probes - Section 13.3. It should be possible to modify the Amplex Red cholesterol assay to continuously measure the activity of cholesterol esterases.

The Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit (A12217), which is discussed in Substrates for Oxidases, Including Amplex Red Kits - Section 10.5 and Probes for Neurotransmitter Receptors - Section 16.2, provides an ultrasensitive method for continuously monitoring acetylcholinesterase activity or for detecting acetylcholine in a fluorescence microplate reader or fluorometer (Figure 16.27, Figure 16.28).

Figure 16.27 Detection of electric eel acetylcholinesterase activity using the Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit (A12217). Each reaction contained 50 µM acetylcholine, 200 µM Amplex Red reagent, 1 U/mL HRP, 0.1 U/mL choline oxidase and the indicated amount of acetylcholinesterase in 1X reaction buffer. Reactions were incubated at room temperature. After 15 and 60 minutes, fluorescence was measured in a fluorescence microplate reader using excitation at 560 ± 10 nm and fluorescence detection at 590 ± 10 nm. The inset shows the sensitivity of the 15 min () and 60 min () assays at low levels of acetylcholinesterase activity (0–13 mU/mL).

Figure 16.28 Detection of acetylcholine using the Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit (A12217). Each reaction contained 200 µM Amplex Red reagent, 1 U/mL HRP, 0.1 U/mL choline oxidase, 0.5 U/mL acetylcholinesterase and the indicated amount of acetylcholine in 1X reaction buffer. Reactions were incubated at room temperature. After 15 and 60 minutes, fluorescence was measured with a fluorescence microplate reader using excitation at 560 ± 10 nm and fluorescence detection at 590 ± 10 nm. The inset shows the sensitivity of the 15 min () and 60 min () assays at low levels of acetylcholine (0–3 µM).

Acetylcholinesterase has also been assayed using the acetylcholinesterase-catalyzed release of thiocholine from acetylthiocholine, followed by detection of the thiol with the essentially nonfluorescent 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM, D346; Thiol-Reactive Probes Excited with Ultraviolet Light - Section 2.3) dye or Ellman's reagent (DTNB, D8451; Thiol-Reactive Probes Excited with Ultraviolet Light - Section 2.3).

In a similar assay, the activity of histone acetyltransferase, which transfers an acetyl group from acetyl coenzyme A to lysine residues in the N-terminal tail of histones, was measured by using CPM to derivatize the thiol-containing coenzyme A (CoASH) product.

Because of the close correlation between its transcript levels and enzymatic activity and the excellent sensitivity of the enzyme assay, the chloramphenicol acetyltransferase (CAT) reporter gene system has proven to be a powerful tool for investigating transcriptional elements in animal and plant cells. Most conventional CAT assays require incubation of cell extracts with radioactive substrates, typically ¹⁴C chloramphenicol or ¹⁴C acetyl CoA, followed by organic extraction and autoradiography or scintillation counting. Molecular Probes' Patented FAST CAT Chloramphenicol Acetyltransferase Assay Kits contain unique BODIPY chloramphenicol fluorescent substrates that take advantage of the exquisite sensitivity of fluorescence techniques, thus eliminating the need for hazardous radiochemicals, film, fluors, scintillation counters and expensive radioactive waste disposal. The original FAST CAT Kit and our FAST CAT Green and Yellow (deoxy) Kits provide detection limits similar to those achieved with conventional radioactive methods and yield results that are easily visualized using a hand-held UV lamp.

FAST CAT Chloramphenicol Acetyltransferase Assay Kit

The green-fluorescent BODIPY FL chloramphenicol substrate in our original FAST CAT Chloramphenicol Acetyltransferase Assay Kit (F2900) has a K_M for purified CAT of 7.4 µM and a V_max of 375 picomoles/unit/minute, values that are similar to those of ¹⁴C-labeled chloramphenicol (K_M = 12 µM and V_max = 120 picomole/unit/minute). To perform the assay, cell extracts are simply incubated with BODIPY FL chloramphenicol and acetyl coenzyme A. After a suitable incubation period, the products and remaining substrate are extracted and separated by thin-layer chromatography (TLC). The brightly fluorescent, well-resolved spots can be immediately visualized with a hand-held UV lamp or quantitated with a laser scanner or CCD camera (). Alternatively, quantitation can be accomplished using a fluorometer or spectrophotometer after a simple extraction. HPLC analysis of the fluorescent products has also been used to further enhance the assay's sensitivity.

These attributes have enabled researchers to use this FAST CAT substrate to measure CAT activity in crude cellular extracts of transfected ovarian granulosa cells. Our FAST CAT Kit has also been employed to study hormonal regulation of prodynorphin gene expression and to measure the rate of hair growth in single follicles of transgenic mice.

Each FAST CAT Chloramphenicol Acetyltransferase Assay Kit (F2900) is available in a 100-test size and includes:

• BODIPY FL chloramphenicol substrate
• Mixture of the 1- and 3-acetyl and 1,3-diacetyl BODIPY FL derivatives, which serve as a reference standard for the fluorescent products
• Detailed protocols (FAST CAT(R) Chloramphenicol Acetyltransferase Assay Kit)

FAST CAT (Deoxy) Chloramphenicol Acetyltransferase Assay Kits

Molecular Probes' two FAST CAT (deoxy) Chloramphenicol Acetyltransferase Assay Kits (F6616, F6617) contain substrates that greatly simplify the quantitation of chloramphenicol acetyltransferase (CAT) activity and extend the linear detection range of Molecular Probes' original FAST CAT assay. The BODIPY FL chloramphenicol substrate in our original FAST CAT kit contains two acetylation sites, only one of which is acetylated by the CAT enzyme. Once the CAT enzyme adds an acetyl group to this position, the acetyl group can be nonenzymatically transferred to the second site, leaving the original position open for another enzymatic acetylation. Therefore, enzyme acetylation of our original BODIPY FL FAST CAT substrate produces three products — one diacetylated and two monoacetylated chloramphenicols — thus complicating the quantitative analysis of CAT gene activity. More importantly, because the nonenzymatic transacetylation is the rate-limiting step, the rate of product accumulation may not accurately reflect CAT activity.

To overcome this limitation, we have modified the original FAST CAT substrate, producing reagents that undergo a single acetylation reaction (Figure 10.71). The green-fluorescent BODIPY FL deoxychloramphenicol and yellow-fluorescent BODIPY 543/569 deoxychloramphenicol substrates in our FAST CAT Green and FAST CAT Yellow (deoxy) Chloramphenicol Acetyltransferase Assay Kits (F6616, F6617) are acetylated at a single position, yielding only one fluorescent product (Figure 10.71). This simplified reaction scheme provides a straightforward and reliable measure of CAT activity and extends the linear detection range of our original FAST CAT assay.

The FAST CAT Green and FAST CAT Yellow (deoxy) Chloramphenicol Acetyltransferase Assay Kits are available in a 100-test size and include:

• BODIPY FL 1-deoxychloramphenicol substrate (in Kit F6616) or BODIPY TMR 1-deoxychloramphenicol substrate (in Kit F6617)
• 3-Acetyl BODIPY FL derivative (in Kit F6616) or BODIPY TMR derivative (in Kit F6617), which serves as a reference standard for the fluorescent product
• Detailed protocols (FAST CAT(R) (deoxy) Chloramphenicol Acetyltransferase Assay Kits)

The CAT substrate in our FAST CAT Green (deoxy) Chloramphenicol Acetyltransferase Assay Kit (F6616) is spectrally identical to the green-fluorescent BODIPY FL chloramphenicol substrate in our original FAST CAT Kit. The FAST CAT Yellow (deoxy) Chloramphenicol Acetyltransferase Assay Kit (F6617) contains a red-orange–fluorescent BODIPY TMR derivative. The availability of two spectrally distinct CAT substrates allows researchers to choose the optimal fluorophore for a particular excitation source or multicolor labeling experiment.

Figure 10.71 The green-fluorescent BODIPY FL 1-deoxychloramphenicol substrate in our FAST CAT Green (deoxy) Chloramphenicol Acetyltransferase Assay Kit (F6616). CAT-mediated acetylation of this substrate and of the BODIPY TMR 1-deoxychloramphenicol in our FAST CAT Yellow (deoxy) Chloramphenicol Acetyltransferase Assay Kit (F6617) results in single fluorescent products because these substrates contain only one hydroxyl group that can be acetylated. In contrast, the BODIPY FL chloramphenicol substrate in our original FAST CAT Kit (F2900) contains a second hydroxyl group at the 1-position (indicated by the labeled arrow). This hydroxyl group undergoes a nonenzymatic transacetylation step, restoring the original hydroxyl for a second acetylation. CAT-mediated acetylation of this chloramphenicol substrate produces three fluorescent products, thus complicating the analysis.

Firefly luciferase (Photinus-luciferin:oxygen 4-oxidoreductase or luciferin 4-monooxygenase, EC 1.13.12.7) produces light by the ATP-dependent oxidation of luciferin (Figure 15.58). The 560 nm chemiluminescence from this reaction peaks within seconds, with light output that is proportional to luciferase concentration when substrates are present in excess. The luc gene, which encodes the 62,000-dalton firefly luciferase, is a popular reporter gene for plants, bacteria and mammalian cells and for monitoring baculovirus gene expression in insects. Chemiluminescent techniques are virtually background-free, making the luc reporter gene ideal for detecting low-level gene expression.

Figure 15.58 Reaction scheme for bioluminescence generation via luciferase-catalyzed conversion of luciferin (L2911, L2912, L2916) to oxyluciferin.

Luciferin

The substrate for firefly luciferase, D-(-)-2-(6'-hydroxy-2'-benzothiazolyl)thiazoline-4-carboxylic acid, commonly known as luciferin, was first isolated by Bitler and McElroy (9 mg from approximately 15,000 fireflies!). In the firefly, spent luciferin (oxyluciferin) is recycled back to luciferin. Molecular Probes is a primary manufacturer of synthetic luciferin (L2911) and its water-soluble sodium (L2912, L22172) and potassium (L2916) salts. The physical properties of these derivatives are identical to those of the natural compound. Our prices for luciferin are significantly lower than those of other suppliers; additional discounts are available for bulk purchases or standing orders.

Typically, luciferase expression is measured by adding the substrates ATP and luciferin to cell lysates and then analyzing light production with a luminometer. As little as 0.02 pg (250,000 molecules) of luciferase can be reliably measured using a standard scintillation counter. Moreover, a CCD-based imaging method of detecting luc gene expression in single cells has been developed.

Caged Luciferin

Although luciferase activity is sometimes measured in living cells, in vivo quantitation appears to be limited by the difficulty in delivering luciferin into intact cells. Molecular Probes' DMNPE-caged luciferin (L7085) readily crosses cell membranes. Once the caged luciferin is inside the cell, active luciferin can be released either instantaneously by a flash of UV light or continuously by the action of endogenous intracellular esterases, which are found in many cell types. This probe should facilitate in vivo luciferase assays in two important ways. First, caged luciferin improves the sensitivity and quantitative analysis of these assays by allowing more efficient delivery of luciferin into intact cells. Second, hydrolysis by intracellular esterases provides a continuous supply of active luciferin, permitting long-term measurements and reducing the need for rapid mixing protocols and costly injection devices. Moreover, DMNPE-caged luciferin may make it easier to follow dynamic changes in gene expression in live cells. Molecular Probes also offers DMNPE-caged ATP (A1049, Photoactivatable Reagents, Including Photoreactive Crosslinkers and Caged Probes—Section 5.3), which can be used in conjunction with DMNPE-caged luciferin (L7085) for this in vivo luciferase assay.

Luciferin–Luciferase Assays for ATP, Anesthetics and Hormones

Luciferin has been used in an exquisitely sensitive and specific ATP assay, which allows the detection of femtomolar quantities of ATP. This bioluminescent ATP assay has been employed to determine cell proliferation and cytotoxicity in both bacteria and mammalian cells. Molecular Probes provides all the reagents needed for this important assay in its ATP Determination Kit (A22066, Detecting Enzymes That Metabolize Phosphates and Polyphosphates—Section 10.3).

Researchers have also adapted the luciferin–luciferase ATP assay system for detecting single base changes in a solid-phase DNA sequencing method. In addition, amphipathic and hydrophobic substances, including certain anesthetics and hormones, compete with luciferin for the hydrophobic site on the luciferase molecule, providing a convenient method to assay subnanomolar concentrations of these substances. A protein A–luciferase fusion protein has been developed that can be used in bioluminescence-based immunoassays.

Coelenterazines for Renilla Luciferase

Coelenterazine and its analogs are substrates for the bioluminescent Renilla luciferase. Molecular Probes offers coelenterazine (C2944) and several synthetic coelenterazine analogs, including coelenterazine cp, f, h, hcp and n (C14260, C6779, C6780, C14261, C6776; Protein-Based Ca2+ Indicators—Section 19.5; Coelenterazines and their properties—Table 19.4). Luciferin and coelenterazine have been used together for dual detection of firefly and Renilla luciferases in live mice. Coelenterazine analogs have been characterized for their effectiveness in measuring Renilla luciferase in both live cells and live animals. Our Coelenterazine Sampler Kit (C6777) contains 25 µg samples of each of our coelenterazine analogs, as well as 25 µg of native coelenterazine. Coelenterazine is readily solubilized in aqueous solutions containing 50 mM hydroxypropyl-β-cyclodextrin.

Fluorocillin Green 495/525 β-Lactamase Substrate

Fluorocillin Green 495/525 substrate (F33952) is a robust reporter for ELISA protocols that employ TEM-1 β-lactamase conjugates. Upon cleavage, Fluorocillin Green 495/525 reagent is converted to a green-fluorescent soluble product (excitation/emission maxima ~495/525 nm). Fluorocillin Green 495/525 reagent has a broad dynamic range of fluorescence signal, is more sensitive than common colorimetric substrates and displays only modest hydrolysis when incubated for extended periods from pH 5.5 to 8.0. Additionally, Fluorocillin Green 495/525 reagent consistently reports β-lactamase activity in the presence of EDTA, many detergents, salts and sodium azide. Fluorocillin Green 495/525 reagent is available as a dry powder, packaged in five vials each containing 100 µg (F33952), and as a component of the SensiFlex ELISA Development Kits (S33853, S33854; Secondary Immunoreagents - Section 7.2).

Fluorocillin Green 345/530 β-Lactamase Substrate

Fluorocillin Green 345/530 β-lactamase substrate (F33951) was developed as a precipitating dye generally compatible with a variety of β-lactamase enzymes and corresponding antibody conjugates. Scientists at Molecular Probes have demonstrated the utility of this reagent in immunohistochemistry applications, and it may also prove useful as a marker for endogenous β-lactamase activity in prokaryotes, possibly detected by microscopy or even flow cytometry. It is important to note, however, that in its precipitated form, Fluorocillin Green 345/530 β-lactamase substrate produces a crystal size incompatible with immunocytochemical analysis. This form of β-lactamase substrate is also incompatible with TEM-1 β-lactamase and its conjugates, but can, for example, be cleaved by P99 β-lactamase.

Resazurin (R12204), which under the name alamarBlue (a trademark of AccuMed International, Inc.) has been reported to be useful for quantitatively measuring cell-mediated cytotoxicity, cell proliferation and mitochondrial metabolic activity in isolated neural tissue, is also a useful substrate for measuring the dehydrogenase activity or a wide variety of dehydrogenase enzymes in vitro. Among the assays reported are the use of resazurin to detect:

• Serum formate using formate dehydrogenase and NAD⁺
  
• Bile acids in human urine, feces and serum using NAD⁺ 3α-hydroxysteroid dehydrogenase
  
• Glucose 6-phosphate dehydrogenase (G6PD) activity
  
• NADH and bile acids with NADH oxidoreductase
  
• Triacylglycerols with glycerol dehydrogenase
  
• Argininosuccinate lyase and NAPDH by a coupled diaphorase–resazurin reaction sequence
  
• Urinary acylcarnitines in an immobilized enzyme reactor

Our extensive bibliography on resazurin (Bibliography for R12204) includes numerous references in which this reagent has been referred to as alamarBlue. The dehydrogenase substrate in our Vybrant Cell Metabolic Assay Kit and LIVE/DEAD Cell Vitality Assay Kit (V23110, L34951; Viability and Cytotoxicity Assay Kits for Diverse Cell Types - Section 15.3) is dodecylresazurin, a more lipophilic version of resazurin. Because this substrate readily penetrates the membranes of live cells and its fluorescent reduction product (dodecylresorufin) is better retained in cells, it is preferred for both microscopy and flow cytometry assays. See Viability and Cytotoxicity Assay Kits for Diverse Cell Types - Section 15.3 for more details.

Glucose 6-phosphate dehydrogenase (G6PD) is a ubiquitous enzyme that is part of the pentose phosphate pathway, and is crucial for cellular antioxidant defenses via its production of NADPH. Our Vybrant Cytotoxicity Assay Kit (V23111) was designed to monitor the release of this cytosolic enzyme from damaged cells into the surrounding medium. However, our method also provides an extremely sensitive and specific assay for G6PD in cell-free extracts. Detection of G6PD is via a two-step enzymatic process that leads to the reduction of resazurin into the red-fluorescent resorufin (Figure 15.36). The resulting signal is proportional to the amount of G6PD released into the cell media, which correlates with the number of dead cells in the sample (Figure 15.37).

Figure 15.37 Detection of dead and dying cells using the Vybrant Cytotoxicity Assay Kit (V23111). Jurkat cells were treated with 10 µM camptothecin for six hours, then assayed for glucose 6-phosphate dehydrogenase release. An untreated control sample is shown for comparison. The fluorescence was measured in a microplate reader (excitation/emission ~530/590 nm). A background of 55 fluorescence units was subtracted from each value.

The Vybrant Cytotoxicity Assay Kit contains all enzymes and substrates needed to measure the activity of G6PD and to detect the release of G6PD from damaged and dying cells. The assay can be completed in less than an hour and is effective with as few as 500 cells per sample. Resorufin, the end product of the G6PD cytotoxicity assay, has absorption and emission maxima at ~571 nm and 585 nm, respectively, placing the fluorescent signal beyond the autofluorescence of most biological samples. In addition, the levels of G6PD in serum commonly used for cell culture are much lower than those of lactate dehydrogenase (LDH), an enzyme often used in similar assays, thus resulting in lower background signals (Figure 15.38). The Vybrant Cytotoxicity Assay Kit (V23111) contains:

• Resazurin (5 vials)
  
• Dimethylsulfoxide (DMSO)
  
• Reaction mixture (diaphorase, glucose 6-phosphate and NADP⁺)
  
• Reaction buffer
  
• Cell lysis buffer
  
• Detailed protocols for the assay (Vybrant(R) Cytotoxicity Assay Kit)