Detectagene blue cmcg lacz gene expression kit

DetectaGene™ Blue CMCG lacZ Gene Expression Kit (D-2921) For Detecting β-Galactosidase Activity in Living Cells ological conditions, however, the fluorescent hydrolysis product(fluorescein) leaks quickly from the lacZ-positive cells after enzymatic cleavage. To retard leakage, the cells must be main- tained in conditions that reduce cell viability prior to β-galacto- To overcome the limitations of these substrates, scientists at Molecular Probes have developed the DetectaGene Blue lacZGene Expression Kit with a unique β-galactosidase substrate thatyields a bright blue fluorescent product with greatly improvedcellular retention. The fluorogenic substrate in our DetectaGeneBlue lacZ Gene Expression Kit is 4-chloromethylcoumarin β-D-galactopyranoside (CMCG, Figure 1). This substrate has been designed to react with intracellular glutathione, a ubiquitous tri-peptide, through a glutathione S-transferase–mediated reaction.
The Escherichia coli β-D-galactosidase gene (lacZ) is an im- In lacZ-positive cells, the CMCG–glutathione adduct is subse- portant reporter gene for detecting the expression of recombinant quently converted to a bright blue fluorescent product. Because genes in animal cells. Once reporter genes are fused with other peptides do not readily cross cellular membranes, the resulting genes or with genomic regulatory elements, the resulting DNA 7-hydroxycoumarin–glutathione adduct is well retained, even constructs can be introduced into cells of interest and the reporter in cells that are kept at 37°C. Molecular Probes’ researchers gene product can then be assayed. In present analytical tech- have found that lacZ-positive CRE BAG 2 cells loaded with niques, transcription from the transfected promoter is monitored 200 µM CMCG are highly fluorescent. Moreover, the CMCG- by RNA analysis or by the detection of an encoded protein prod- stained cells were found to be fluorescent even after incubation at uct. Typically, reporter genes encode enzymes not ordinarily found in the type of cell being studied, and their unique activity The DetectaGene Blue Kit also includes stock solutions of is monitored to determine the degree of transcription of the for- phenylethyl β-D-thiogalactopyranoside (PETG), chloroquine eign genetic material. The E. coli lacZ gene has been extensively diphosphate and propidium iodide. PETG is a competitive in- studied and utilized for this purpose.
hibitor of β-galactosidase that can be added to terminate reac- 5-Bromo-4-chloro-3-indolyl galactopyranoside (X-gal) is used tions prior to analysis. Chloroquine may be used to lower for detection of genes sequentially fused in frame with the lacZ lysosomal pH and thereby inhibit the interfering endogenous gene. When X-gal is cleaved, an intensely blue halogenated in- lysosomal β-galactosidase activity present in some mammalian doxyl derivative is formed that is effective for visual identifica- cells. Propidium iodide is useful for identifying dead cells in the tion of transformed cells. However, the cleavage product of population; this dye permeates damaged plasma membranes of X-gal is nonfluorescent and is toxic to viable cells and therefore dead cells and results in a red fluorescent nuclear stain.
not useful for fluorescence-activated cell sorting analysis. Forthis reason, the fluorescent β-galactosidase substrate, fluoresceindi-β-D-galactopyranoside (FDG), has been used for a highly sen- sitive flow cytometric β-galactosidase assay.1,2 Under physi- $ DetectaGene Blue substrate reagent (Component A),
100 µL of 10 mM 4-chloromethylcoumarin β-D-galacto- pyranoside (CMCG) in 1:1 (v/v) water/dimethylsulfoxide(DMSO).
$ PETG (Component B), 1 mL of 50 mM phenylethyl β-D-thio-
$ Chloroquine (Component C), 1 mL of 30 mM chloroquine
$ Propidium iodide (Component D), 1 mL of 150 µM
Figure 1. Structure of CMCG.
DetectaGene™ Blue CMCG lacZ Gene Expression Kit Using the protocol provided, this kit provides sufficient reagents 1.4 Immediately before use, prepare 100 µL of 200 µM CMCG
for at least 50 tests when loading cells in suspension and 25 tests substrate working solution in deionized water from the 10 mM stock solution (Component A) (notes D and E). Warm the solu-
tion at 37°C for about 10 minutes.
The stock CMCG reagent is stable for several months if 1.5 Combine 100 µL of pre-warmed CMCG substrate working
stored frozen. To reduce decomposition of this reagent during solution with 100 µL of pre-warmed cells suspended from step freezing and thawing, we recommend that you divide the reagent 1.3. Mix rapidly and thoroughly. Return the sample to the 37°C into several small aliquots and store at -20°C. The CMCG re- water bath for 2 minutes. Note: The optimal working concen-
agent can be diluted fivefold with distilled water to facilitate tration of CMCG substrate must be determined experimentally.
this distribution. Do not keep the CMCG working solution at The recommended 200 µM working concentration suggested elevated temperatures for extended periods, as spontaneous may have to be varied based on the method of loading (note E)
hydrolysis may occur. Note: The presence of a pronounced
and the level of β-galactosidase activity in cells.
blue-green color in the CMCG reagent or observation of an un-usually high fluorescent background in the cells may indicate 1.6 Stop the CMCG loading at the end of 2 minutes by adding
1.8 mL of Staining Medium to the 200 µL volume of CMCG and The other reagents included in this kit are also stable for sev- eral months when stored frozen at -20°C. Minimize exposure tolight.
1.7 Wash the cells by centrifugation and resuspend them in
2.0 mL of Staining Medium. If desired, 1.5 µM propidium
iodide may be included in the Staining Medium to facilitate the
identification of dead cells (note F).
The DetectaGene Blue CMCG lacZ Gene Expression Kit 1.8 Keep cells under normal culture conditions for 30 minutes
can be used for either fluorescence imaging or flow cytometric to allow for complete turnover of the substrate prior to flow cyto- analysis of β-galactosidase–containing cells. Detection by flow metric analysis. Note: At any point after the termination of
cytometry using the DetectaGene Blue CMCG substrate requires loading, you may inhibit continuing intracellular hydrolysis of a flow cytometer that can provide UV excitation. The following the substrate by treatment with PETG (see note G).
protocol includes the basic methodology for preparing adherentcells or cells in suspension, staining them with CMCG and detecting fluorescence in a fluorescence microscope or flow cy- 2.1 Grow cells on coverslips according to normal tissue culture
tometer. Also described are methods for using the competitive procedures. Use cells at a 40% to 70% confluency for best re- inhibitor, PETG, to slow or completely block β-galactosidase sults (note A). If inhibition of endogenous β-galactosidase
activity and methods for using chloroquine to lower the back- is desired, prepare Staining Medium with 1 mM chloroquine ground from endogenous lysosomal β-galactosidase activity, diphosphate (freshly diluted from the 30 mM stock, (Compo- which is present in some cells. This protocol should serve as a nent C); concentrations greater than 1 mM may be deleterious to guideline and may require modification based on specific experi- cells 3 (note C).
2.2 Immediately before use, dilute the CMCG substrate stock
reagent (Component A) to 400 µM using a 1:1 mixture of deion- Make up 10 mL of Staining Medium. A typical staining ized water and Staining Medium. Warm the substrate solution at medium is phosphate-buffered saline (PBS), 4% (v/v) fetal calf 37°C for 10 minutes. A 100 µL volume will be used for each 2.3 Rinse cells once with a physiological saline solution such as
1.1 Centrifuge cells to obtain a cell pellet and aspirate the super-
Hank’s balanced salt solution or PBS.
natant (note A). Resuspend cells in Staining Medium and draw
through a pipet to obtain a single cell suspension. Filter out any
2.4 Place coverslip with adherent cells in a petri dish. Apply
cell clumps with a nylon screen. Centrifuge cells again and re- 100 µL of substrate solution to the coverslip and incubate the sample at room temperature for 1 minute.
1.2 Resuspend the cells in Staining Medium to approximately
2.5 Stop the CMCG loading by flooding the petri dish with Stain-
107 cells/mL (note B) and pipet 100 µL into a centrifuge tube.
ing Medium. If desired, 1.5 µM propidium iodide may be included If inhibition of endogenous β-galactosidase is desired, prepare in the Staining Medium to facilitate the identification of dead cells Staining Medium with 1 mM chloroquine diphosphate (freshly (note F). Note: Do not remove the substrate solution before flood-
diluted from the 30 mM stock, (Component C)); concentrations ing with medium as this will often wash away many of the cells.
greater than 1 mM may be deleterious to cells 3 (note C). Pro-
ceed to step 1.3 immediately, or put cells on ice.
2.6 Return the cells to the 37°C incubator and allow the cells to
recover for 1–3 hours. Note: At any point after the termination
1.3 Pre-warm the tube containing 100 µL of cells in a 37°C
of loading, you may inhibit further intracellular hydrolysis of the water bath for 10 minutes, or for 30 minutes if inhibiting endog- substrate by treatment with PETG (see note G).
enous β-galactosidase with chloroquine diphosphate.
DetectaGene™ Blue CMCG lacZ Gene Expression Kit 2.7 Mount the cells in staining medium on a slide. Seal and
[D] For bacterial cells or yeast, the cell wall restricts the swelling
view immediately. For flow cytometric assay, treat adherent cells induced by osmotic loading, thus preventing CMCG entry. Brief with trypsin in phosphate buffer until they can be removed from (1–3 minute) hypertonic shrinking of the cell membrane within the plate by gentle agitation. Afterwards, remove the trypsin by the wall, followed by a 2-minute hypotonic loading of CMCG washing in Staining Medium. Centrifuge the cell suspension, can correct this difficulty with entry.
aspirate off the supernatant and resuspend the cells in StainingMedium.
[E] The loading procedure described in steps 1.5 and 2.4 sub-
jects cells to hypotonic shock in order to facilitate substrate entry.
This treatment may not be necessary for some cell types. For
loading under isotonic conditions, prepare the CMCG workingsolution in staining medium instead of distilled water and in- crease the incubation time from 2 to about 30 minutes.
Set up and calibrate the flow cytometer to detect coumarin, propidium iodide and forward scatter according to standard pro- [F] Propidium iodide is impermeant to the plasma membrane
cedures.4 Use unstained cells of the same type you are analyzing and selectively labels the nuclei of dead cells with red fluores- to set the background autofluorescence compensation 5 (note H).
[G] Competitive inhibition of β-galactosidase by PETG (Compo-
Fluorescence is detected using standard coumarin or DAPI nent B) can be used to terminate CMCG turnover prior to analy- sis. After terminating CMCG loading select a time intervalbetween zero and 60 minutes (zero time for cells with high lacZexpression levels, 60 minutes for cells with low lacZ expression levels) and add an aliquot of the 50 mM PETG stock reagent toyield a final PETG concentration of 1 mM. Mix thoroughly.
[A] Keep cells as healthy as possible. Endogenous lysosomal
PETG is a competitive, reversible inhibitor of E. coli β-galac- β-galactosidase activity increases dramatically if cells are tosidase in mammalian cells. It is hydrophobic and can readily abused or allowed to reach confluency (see note C on inhibition
cross the cell membrane to inhibit β-galactosidase. Because it of endogenous β-galactosidase activity with chloroquine diphos- has a low K (3 × 10-6 M), very little PETG is required to inhibit the reaction. In addition, PETG is not hydrolyzed by the enzyme,which simplifies its influence on the kinetics.
[B] Staining results are not critically dependent on cell concen-
tration. Staining patterns are essentially the same using cell con-
[H] Some endogenous constituents of cells give rise to broad
centrations ranging from 105 cells/mL to 5 × 107 cells/mL.
bandwidth autofluorescence when excited by the argon laser. Itis essential to compensate for autofluorescence in order to accu- [C] Some mammalian cells have endogenous lysosomal β-galac-
rately measure low levels of β-galactosidase activity. Correction tosidase that can interfere with accurate measurement of lacZ for the autofluorescence component of the emission signal is expression. The endogenous activity can be selectively de- typically based on the proportionality of measured autofluores- pressed by pre-incubating cells with the weak base, chloroquine cence at one wavelength to that at another wavelength.5 References
1. Proc Natl Acad Sci USA 85, 2603 (1988); 2. Cytometry 12, 291 (1991); 3. Exp Cell Res 136, 327 (1981); 4. Parks, D.R. et al. in The Handbook of Experi-
mental Immunology 4th edition, D.M. Wier, Ed., Blackwell, Edinburgh (1986) pp 29.1–29.21; 5. Cytometry 8, 114 (1988).
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Cat # DetectaGeneTM Blue CMCG lacZ Gene Expression Kit .
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DetectaGene™ Blue CMCG lacZ Gene Expression Kit

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