ab189815 FACS Blue LacZ beta Galactosidase detection kit Instructions for Use For the detection of beta-galactosidase using Enzyme or FACS Assay This product is for research use only and is not intended for diagnostic use. Version 2 Last Updated 2 April 2015
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Table of Contents 1. Introduction 3 2. Protocol Summary 5 3. Kit Contents 7 4. Storage and Handling 7 5. Additional Materials Required 8 6. Assay Protocol 9 7. Troubleshooting 19 2
1. Introduction One of the most common reporter genes used in molecular biology applications is the E. coli lacz gene that codes for an active subunit of β-galactosidase in vivo. Since this enzyme is generally absent in normal mammalian, yeast, some bacterial and even plant cells, it can be detected at very low levels, and since the enzyme has a wide substrate specificity, monitoring lacz expression (and therefore coexpressed genes or promoter efficiency) has become routine to the point of detection of as few as 5 copies of β-galactosidase per cell by FACS analysis. Although chromogenic assays of β-galactosidase activity (i.e. X-Gal) have use, application of the fluorogenic substrate 3-carboxyumbelliferyI β-d-galactopyranoside (CUG) (blue fluorescence) combined with Fluorescence Activated Cell Sorting (FACS) analysis has been shown to be several orders of magnitude more sensitive. In addition, because of its high water solubility and detection limits, the CUG substrate has found extensive use in automated ELISA type assay systems. This assay system is especially useful for dual-labeling experiments where the cells to be analyzed are also labeled with a fluorescein based probe (FITC-labeled antibody, fluorescein-based substrate, etc.). 3
Abcam s FACS Blue LacZ beta Galactosidase detection kit (ab189815) provides reagents and protocol to perform up to 500 tests (10 x 96 well microtitre plates). Emission: 460 nm. Excitation: 390 nm. 4
2. Protocol Summary Enzyme Assay (microtitre plate format) Pipette serial dilutions of sample into 96 well plate Add Reaction Buffer Add 10 mm Substrate Reagent Incubate Add Stop Buffer and read fluorescence 5
FACS Assay Harvest cells Spin cells down to form pellet Add warm 0.5 mm staining media Incubate Add ice-cold media and read fluorescence 6
3. Kit Contents Components Amount Storage Substrate Reagent 0.5 ml Store at or below -20 C Light sensitive Reference Standard 0.5 ml Store at or below -20 C Light sensitive Inhibitor 1 ml Store at or below -20 C 4. Storage and Handling Fluorescent reagents and fluorogenic substrates should be handled with care, kept cold (ice-bath) when not in use, and stored frozen (-20 C). In case of contact with skin or eyes wash thoroughly with soap and cold water. High background fluorescence readings for blank samples will indicate decomposition. Avoid exposure of Substrate Reagent and Reference Standard to light. 7
5. Additional Materials Required 96 or 384-well black wall/clear bottomed microplates Fluorescence microplate reader Parafilm CO 2 incubator Flow cytometer Water bath Distilled water Absolute Methanol 100 mm sodium phosphate buffer, ph 7.0 1 mm MgCl 2 10 mm β-mercaptoethanol 0.1% Triton X-100 500 mm glycine 10 mm EDTA 10 M NaOH 8
6. Assay Protocol A. Protocol for Plate Reading 1. Prepare Substrate Reagent Add 0.5 ml of 50 mm in distilled water. Dilute with 2.0 ml of the Reaction Buffer to prepare the reagent for use in the Enzyme Assay below. 2. Prepare Reference Standard Add 0.5 ml of 20 µm in absolute methanol. Dilute with Stop Buffer for spectrometer standardization as outlined in FACS protocol below. 3. Prepare Inhibitor Add 0.1 ml of 30 mm in distilled water. 4. Prepare Buffer Solutions (not provided) Prepare Reaction Buffer containing 100 mm sodium phosphate buffer, ph 7.0 with 1 mm MgCl 2, 10 mm β-mercaptoethanol and 0.1% Triton X-100. Prepare Stop Buffer containing 500 mm glycine and 10 mm EDTA. Adjust the ph to 12.0 with concentrated NaOH (10 M). 9
5. Run Enzyme Assay It is recommended that a calibration curve be prepared using known concentrations of purified β-galactosidase enzyme in the approximate concentration range of the unknown analyte. Since the conversion of the fluorogenic substrate (CUG) releases the fluorophore 7-hydroxycoumarin-3 carboxylic acid (CU), the emission of this highly fluorescent product is monitored at 460 nm using excitation at 390 nm. To normalize data, each enzyme reaction is terminated at exactly the same time (20 min.) using a stop buffer of high ph (12.0). The enzyme assay has a typical working range from about 1-1000 picograms. Adjust enzyme concentrations accordingly. The sample containing cell lysate, purified enzyme, or cell suspension is pipetted into individual microtiter plate wells with serial dilutions in duplicate for each concentration and for each cell/tissue sample (20-50 µl/well). Include two wells for blanks (20-50 µl/well reaction buffer). Add 100 µl Reaction Buffer to each well. Incubate/mix for a few minutes to make sure the reaction system is homogeneous. 10
Add 50 µl 10 mm Substrate Reagent to each well. Mix thoroughly by manual agitation. Note: Lower concentrations of Substrate Reagent (1-5 mm) are routinely used for lower enzyme concentrations. Incubate for 20 min, at a fixed temperature (normally 25 C). Note: If lower substrate concentrations are used, incubation times may need to be adjusted proportionally. Add 100 µl Stop Buffer to each well. Wait for 10 min. Emission readings can now be made at any time up to 3 hours after stopping the reaction. Store unread plates (4 C) covered by parafilm or plastic wrap if they are not to be read immediately. Read fluorescence at 460 nm using an appropriate excitation filter for excitation at 390 nm. Use Reference Standard for optimizing spectrometer conditions. Note: A 1:50 dilution with stop buffer will usually be sufficient. Subtract fluorescence from the blank well(s) from each sample well. Average the readings of duplicate samples. Generate a calibration curve using purified enzyme samples by plotting normalized fluorescence vs. β-galactosidase concentration (log-log). 11
Using the calibration curve from above, determine the concentration of β-galactosidase in samples by comparison, and extrapolate this data to determine concentration of the enzyme in the original cell/tissue suspension. 12
B. Protocol for Flow Cytometry It is recommended that measurements be made in duplicate, if possible, and that the approximate concentration range of the fluorescent probe be adjusted for optimum signal and sensitivity. Previous studies have indicated that the labeling of cells is virtually independent of the initial fluorescent probe concentration in the range of about 100 pm - 2 mm. Since staining may be somewhat time dependent, a time course for the experiments should also be generated for initial trials. The emission of the highly fluorescent product carboxyumbelliferone is monitored at 445 nm using excitation at 396 nm. The user is asked to consult with the manufacturer (or instrument manual) for the particular instrument in use for appropriate filter set(s) needed for monitoring at these wavelengths. Typical epifluorescence microscopic analysis is performed using an excitation filter, a dichroic filter and emission filter for coumarin (AMC) fluorescence. For flow cytometric analysis, the FACS instrument is typically equipped with bandpass filter for monitoring coumarin fluorescence. To normalize data, each cell suspension or plate is monitored at exactly the same time (20 minutes) after equilibration with the probe. The product-forming rate is dependent on the concentration of incorporated probe in the cell, but with concentrations of the probe above approximately 2 μm, enzyme kinetics are typically independent of initial concentration. The working range of the assay 13
will need to be determined for each individual experiment. Adjust working concentrations accordingly. A blank prepared with all reagents, substituting corresponding lacz-cells should be run in parallel if possible. Alternately, a blank prepared with all reagents except cells (substituting water or media) can be run to determine background fluorescence readings for each experiment. 1. Prepare Substrate Reagent Add 0.5 ml of 50 mm in distilled water. Dilute with 2.0 ml of the Reaction Buffer to prepare the reagent for use in the Enzyme Assay below. 2. Prepare Reference Standard Add 0.5 ml of 20 µm in absolute methanol. Dilute with Stop Buffer for spectrometer standardization as outlined in FACS protocol below. 3. Prepare Inhibitor Add 0.1 ml of 30 mm in distilled water = 1ml. 4. Prepare Buffer Solutions (not provided) Prepare Reaction Buffer containing 100 mm sodium phosphate buffer, ph 7.0 with 1 mm MgCl 2, 10 mm β- mercaptoethanol and 0.1% Triton X-100. 14
Prepare Stop Buffer containing 500 mm glycine and 10 mm EDTA. Adjust the ph to 12.0 with concentrated NaOH (10 M). 5. Run FACS Assay When using suspension cells, such as bone marrow cells freshly isolated, it has been shown that incubation with CUG for 4-10 min at 37 C followed by cooling on ice 20 minutes prior to FACS analysis gave optimal staining results. Your results may vary. Harvest healthy cells expressing the lacz gene from exponential growing cultures. Note: unhealthy cells or those grown to confluence often exhibit high background staining due to endogenous galactosidase activity or low ph conditions. Trypsinize adherent cells by standard protocols. Spin down cells at 200 x g for 5 minutes to form a cell pellet. Carefully remove supernatant without disturbing cell pellet. Warm cell pellet at 37 C in a water bath for 10 minutes. 15
Meanwhile, prepare staining solution by diluting the Substrate Reagent 1:100 in ice-cold serum-free medium. This will make a 0.5 mm solution. 200 µl of staining media will be needed per sample, plus a bit more for pipetting error. For example, if you have 4 samples, add 10 µl of Substrate Reagent to 990 µl ice-cold serum-free medium. Note: Medium is whatever medium your cells are adapted to, such as MEM, McCoys, DMEM, etc. Warm staining media at 37 C. When cell pellet and staining media are both warm, add 200 µl of staining media to each cell pellet. Note: An unstained cell blank is highly recommended. Mix thoroughly via gentle pipetting. Incubate at 37 C in a water bath for 20 minutes. Terminate dye loading by addition of 1.8 ml ice-cold media containing serum. Note: As the reaction is time-dependent, the reaction time for different samples should be as close to the same length of time as possible. Keep stained cells on ice until analysis. 16
Read fluorescence at 460 nm using an appropriate excitation filter for excitation at 390 nm. Use Reference Standard for optimizing spectrometer conditions. In case high levels of endogenous β-galactosidase are noticed, cells are re-suspended in media at approximately 107 cells per ml containing 300 μm Inhibitor. After incubating at 37ºC for 20 min., the cells are centrifuged and staining is continued as described earlier. For cells with high lacz β-galactosidase activity, CUG loading can be terminated by adding ice-cold media containing the reversible β-galactosidase inhibitor phenylethyl thiogalactoside (PETG) at 1 mm concentration (40 µl of 50 mm PETG/1.8 ml media). 17
6. Quick Test Assay (microtitre plate format) The following is a quick to set up assay that can be used to determine if cells show lacz activity, perhaps to check for transfection efficiency, or in other situations whenever the question is if lacz is present in the cells. The assay can be performed after routine sub culturing of cell lines with as little as 5000 cells, e.g. those remaining after seeding of new vessels. The sample containing cell suspension is pipetted into individual microtiter plate wells in duplicate for each cell/tissue sample (150 µl/well). Include two wells for blanks (150 µl/well of fresh media, no cells). Add 50 µl 10 mm Substrate Reagent to each well. Mix thoroughly. Read fluorescence at 460 nm using an appropriate excitation filter for excitation at 390 nm at regular intervals (e.g. 10 minutes) for approximately 2 hours. Use Reference Standard for optimizing spectrometer conditions. Subtract fluorescence from the blank well(s) from each sample well. Average the readings of duplicate samples. Graphs can then be plotted of increase in fluorescence over time. 18
7. Troubleshooting 1. How long will the Substrate Reagent remain stable? CUG is more stable than FDG for these assays. Still, some care should be exercised when using the kit. The CUG substrate should be stable in solution for at least several hours and even several days, if kept cold. Acidic or basic buffers will, of course hasten the decomposition. Evidence of decomposition can be monitored by running blank (non-lacz cells) at the beginning and end of your assays. 2. Is it possible to put the CU labeled cells in culture, or is the labeling reaction lethal? The CUG and the carboxyumbelliferone (fluorescent product) are not toxic, and you should be able to re-culture your cells after sorting. Keep in mind that some cell toxicity may occur just because of the many manipulations in the FACS sorting, but the staining should not affect viability more than another other type of staining protocol. 19
For further technical questions please do not hesitate to contact us by email (technical@abcam.com) or phone (select contact us on www.abcam.com for the phone number for your region). 20
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