Cell-Based GPCR Reporter Assays - Substrates

LiveBLAzer™ FRET-B/G Loading Kit with CCF4-AM

Background:
GeneBLAzer® cell-based assays utilize the membrane-permeant ester forms (CCF2-AM and CCF4-AM) of the negatively charged fluorescent beta-lactamase substrates, CCF2 and CCF4. These lipophilic esters readily enter the cell, where cleavage by endogeneous cytoplasmic esterases rapidly converts them into their negatively charged forms, thereby trapping them in the cytosol.

Detection of GeneBLAzer® assays is FRET-based. Each substrate is labeled with two fluorophores that form an efficient FRET pair. In the absence of beta-lactamase activity, exciting the coumarin at 409 nm in the intact CCF2 molecule results in FRET to the fluorescein, which emits a green fluorescence signal at 518 nm (Figure 1). In the presence of beta-lactamase activity, however, cleavage of CCF2 spatially separates the two dyes and disrupts FRET, so that exciting the coumarin at 409 nm now produces a blue fluorescence signal at 447 nm. This blue signal can be readily observed under a microscope and can also be detected as an increase in the blue channel readout on fluorescent microplate readers.

The CCF2-AM and CCF4-AM substrates are essential assay components for the GeneBLAzer® platform. These substrates are fully compatible with flow cytometry, speeding the time to clone selection. Ratiometric analysis of the blue and green signals reduces well-to-well variation due to differences in cell numbers and substrate loading, leading to high Z´-factor values and low coefficients of variation (CVs).

CCF2-AM and CCF4-AM differ by two carbons in the bridge linking the coumarin moiety to the lactam ring. Both are in the membrane-permeable, esterified forms, and can be used for assays in intact cells. CCF4-AM has better solubility properties (soluble for >24 hours) than CCF2-AM and is thus best suited for screening applications. In addition, CCF4-AM has slightly better FRET and thus slightly lower background than CCF2-AM.

CCF2-FA is essentially the CCF2 substrate without the esters found in the AM version. CCF2-FA is de-esterified and used in cell lysate applications, bypassing loading across the cell membrane and de-esterification steps. Cell lysates are the preferred method for applications using cells that contain a cell wall. CCF2-FA can also be used as a control to acquire the excitation and emission spectra for CCF2-AM and CCF4-AM.

LyticBLAzer™-FRET B/G Kit, 10X

LyticBLAzer™ -FRET B/G Substrate Kits provides a ratiometric, end-point method of analyzing beta-lactamase activity in cell lysates. The LyticBLAzer™ -FRET B/G Reagent combines the components for lysis and detection in a single reagent addition, resulting in a very simple assay. Use the LyticBLAzer™ on top-read or bottom-read instrumentation. LyticBLAzer™ Substrate Kits are available in homogeneous as well as non-homogeneous formats (Figure 1), each optimized for a different assay condition.

LyticBLAzer™ FRET B/G Substrate Kit Advantages:

• Ratiometric blue:green reporter readout reduces false hits by minimizing experimental noise
• Integrated lysis and detection components enable use with top-read or bottom read fluorescence instrumentation
• Endpoint analysis and stable fluorescence yield longer read times

LyticBLAzer™ -BODIPY® FL Substrate Kit:
LyticBLAzer™ -BODIPY® FL Substrate Kits provides an end-point, non-ratiometric method of analyzing beta-lactamase activity in cell lysates. The LyticBLAzer™ -BODIPY® FL Reagent combines the components for lysis and detection in a single reagent addition, resulting in a very simple assay. Use the LyticBLAzer™ -BODIPY® FL Assays on top-read or bottom-read instrumentation. LyticBLAzer™ -BODIPY® FL Substrate Kits are available in homogeneous as well as non-homogeneous formats (Figure 1), each optimized for a different assay condition.

LyticBLAzer™ -BODIPY® FL Substrate Kit Advantages:
• BODIPY® fluorophore excitation avoids fluorescence interference caused by sample contaminants such as dust, lint, etc.
• Standard excitation and emission wavelengths are universally adaptable to all fluorescence instrumentation.
• Integrated lysis and detection components enable use with top-read or bottom read fluorescence instrumentation.
• Endpoint analysis and stable fluorescence yield longer read times

CCF4-AM

CCF4-AM, our LiveBLAzer™ substrate, is a lipophilic, esterified form of the CCF4 substrate which allows it to readily enter cells. Upon entry, cleavage by endogenous cytoplasmic esterases rapidly converts CCF4-AM into its negatively charged form, CCF4, which is retained in the cytosol. CCF4 is a Fluorescence Resonance Energy Transfer (FRET) substrate which consists of a cephalosporin core linking 7-hydroxycoumarin to fluorescein. This product is to be used as a FRET-based substrate for β-lactamase as a sensitive reporter of mammalian gene expression in the GeneBLAzer® technology.

LiveBLAzer™ FRET-B/G Loading Kit with CCF2-AM

Background:
GeneBLAzer® cell-based assays utilize the membrane-permeant ester forms (CCF2-AM and CCF4-AM) of the negatively charged fluorescent beta-lactamase substrates, CCF2 and CCF4. These lipophilic esters readily enter the cell, where cleavage by endogeneous cytoplasmic esterases rapidly converts them into their negatively charged forms, thereby trapping them in the cytosol.

Detection of GeneBLAzer® assays is FRET-based. Each substrate is labeled with two fluorophores that form an efficient FRET pair. In the absence of beta-lactamase activity, exciting the coumarin at 409 nm in the intact CCF2 molecule results in FRET to the fluorescein, which emits a green fluorescence signal at 518 nm (Figure 1). In the presence of beta-lactamase activity, however, cleavage of CCF2 spatially separates the two dyes and disrupts FRET, so that exciting the coumarin at 409 nm now produces a blue fluorescence signal at 447 nm. This blue signal can be readily observed under a microscope and can also be detected as an increase in the blue channel readout on fluorescent
microplate readers.

The CCF2-AM and CCF4-AM substrates are essential assay components for the GeneBLAzer® platform. These substrates are fully compatible with flow cytometry, speeding the time to clone selection. Ratiometric analysis of the blue and green signals reduces well-to-well variation due to differences in cell numbers and substrate loading, leading to high Z´-factor values and low coefficients of variation (CVs).

CCF2-AM and CCF4-AM differ by two carbons in the bridge linking the coumarin moiety to the lactam ring. Both are in the membrane-permeable, esterified forms, and can be used for assays in intact cells. CCF4-AM has better solubility properties (soluble for >24 hours) than CCF2-AM and is thus best suited for screening applications. In addition, CCF4-AM has slightly better FRET and thus slightly lower background than CCF2-AM.

CCF2-FA is essentially the CCF2 substrate without the esters found in the AM version. CCF2-FA is de-esterified and used in cell lysate applications, bypassing loading across the cell membrane and de-esterification steps. Cell lysates are the preferred method for applications using cells that contain a cell wall. CCF2-FA can also be used as a control to acquire the excitation and emission spectra for CCF2-AM and CCF4-AM.

ToxBLAzer™ DualScreen Kit

ToxBLAzer™ DualScreen Kit provide the key advantage of combining the ratiometric reporter gene readout of beta-lactamase with a cytotoxicity readout in the same assay. Thus, ToxBLAzer™ DualScreen yields reporter readouts that normalize cytotoxic cellular responses, which is a necessity in screening applications which down regulate transcription. The ToxBLAzer™ substrate contains both the LiveBLAzer™-FRET B/G substrate and a proprietary cytotoxicity indicator. Both compounds readily enter the cell, where endogenous esterases rapidly convert them into fluorescent compounds Esterase activity and an intact cell membrane are required for detection of the indicator. The cytotoxicity indicator's fluorescence is red-shifted to ensure compatibility with the blue and green emissions of the LiveBLAzer™-FRET B/G substrate, allowing detection of both reporter gene activity and cytotoxicity in the same assay sample.

Advantages of ToxBLAzer™ DualScreen Kits:

• Multiplexed readout results in rapid identification of false positives due to toxicity or lack of viable cells, thereby improving hit-picking efficiency
• Ratiometric blue:green reporter readout reduces false hits by minimizing experimental noise
• Single reagent addition eliminates need for follow up cytotoxicity assays, thereby increasing throughput while reducing sample and test compound consumption

LyticBLAzer™-FRET B/G Homogeneous Kit

LyticBLAzer™ -FRET B/G Substrate Kits provides a ratiometric, end-point method of analyzing beta-lactamase activity in cell lysates. The LyticBLAzer™ -FRET B/G Reagent combines the components for lysis and detection in a single reagent addition, resulting in a very simple assay. Use the LyticBLAzer™ on top-read or bottom-read instrumentation. LyticBLAzer™ Substrate Kits are available in homogeneous as well as non-homogeneous formats (Figure 1), each optimized for a different assay condition.

LyticBLAzer™ FRET B/G Substrate Kit Advantages:

• Ratiometric blue:green reporter readout reduces false hits by minimizing experimental noise
• Integrated lysis and detection components enable use with top-read or bottom read fluorescence instrumentation
• Endpoint analysis and stable fluorescence yield longer read times

LyticBLAzer™ -BODIPY® FL Substrate Kit:
LyticBLAzer™ -BODIPY® FL Substrate Kits provides an end-point, non-ratiometric method of analyzing beta-lactamase activity in cell lysates. The LyticBLAzer™ -BODIPY® FL Reagent combines the components for lysis and detection in a single reagent addition, resulting in a very simple assay. Use the LyticBLAzer™ -BODIPY® FL Assays on top-read or bottom-read instrumentation. LyticBLAzer™ -BODIPY® FL Substrate Kits are available in homogeneous as well as non-homogeneous formats (Figure 1), each optimized for a different assay condition.

LyticBLAzer™ -BODIPY® FL Substrate Kit Advantages:
• BODIPY® fluorophore excitation avoids fluorescence interference caused by sample contaminants such as dust, lint, etc.
• Standard excitation and emission wavelengths are universally adaptable to all fluorescence instrumentation.
• Integrated lysis and detection components enable use with top-read or bottom read fluorescence instrumentation.
• Endpoint analysis and stable fluorescence yield longer read times

CCF2-FA (free acid)

CCF2-FA (Free Acid) is a fluorescence resonance energy transfer (FRET) substrate that consists of a cephalosporin core linking B7-hydroxycoumarin to fluorescein. This product is intended for use as a beta-lactamase substrate in a GeneBLAzer® cell-based assay.

GeneBLAzer® cell-based assays typically utilize the membrane-permeant ester forms (CCF2-AM and CCF4-AM) of the negatively charged fluorescent beta-lactamase substrates, CCF2 and CCF4. These lipophilic esters readily enter the cell, where cleavage by endogeneous cytoplasmic esterases rapidly converts them into their negatively charged forms, thereby trapping them in the cytosol.

CCF2-FA is essentially the CCF2 substrate without the esters found in the AM version. CCF2-FA is de-esterified and used in cell lysate applications, bypassing loading across the cell membrane and de-esterification steps. Cell lysates are the preferred method for applications using cells that contain a cell wall. CCF2-FA can also be used as a control to acquire the excitation and emission spectra for CCF2-AM and CCF4-AM.

CCF2-AM

CCF2-AM, our LiveBLAzer™ substrate, is a lipophilic, esterified form of the CCF2 substrate which allows it to readily enter cells. Upon entry, cleavage by endogenous cytoplasmic esterases rapidly converts CCF2-AM into its negatively charged form, CCF2 or CCF4, which is retained in the cytosol. CCF2 is a Fluorescence Resonance Energy Transfer (FRET) substrate which consists of a cephalosporin core linking 7-hydroxycoumarin to fluorescein. This product is to be used as a FRET-based substrate for β-lactamase as a sensitive reporter of mammalian gene expression in the GeneBLAzer® technology.