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219346 Cathepsin B Detection Kit

Cathepsin B Detection Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
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      Overview

      Replacement Information

      Key Spec Table

      Detection Methods
      Fluorescence
      Description
      Overview

      This product has been discontinued.





      Detects cathepsin B using a cell-permeable cresyl violet labeled cathepsin B substrate, which generates red fluorescence upon cleavage. This is kit is also suitable for detecting cathepsin B activity in whole cells.
      Catalogue Number219346
      Brand Family Calbiochem®
      Materials Required but Not Delivered• Cultured cells with media
      • Reagents to induce apoptosis or other experimental condition
      • 15 ml polypropylene centrifuge tube (1 per sample)
      • Amber vials or polypropylene tubes for storage of 260X concentrate aliquots at -20°C
      • Slides and coverslips
      • Hemocytometer
      • Clinical centrifuge at 200 X g
      • 37°C CO2 incubator
      • Pipette(s) capable of dispensing at 10, 50, 200, 300 µl, and 1 ml
      • ddH2O, up to 2 ml needed
      • Phosphate buffered saline (PBS) pH 7.4, up to 100 ml needed
      • Dimethyl sulfoxide (DMSO)
      • Trypsin-EDTA
      • Ice buckets or a refrigerator to maintain cells at 4°C
      • Fluorescence microscope with appropriate filters: excitation 550 nm, emission >610 nm for Cathepsin B fluorogenic substrate; excitation at 480 nm and emission at >540 nm for AO; and if Hoechst is used, a UV-filter with excitation at 365 nm, emission at 480 nm.
      References
      ReferencesBoonacker E., and Van Noorden, C.J.F. 2001. J. Histochem. Cytochem. 49, 1473.
      Bank U., et al. 2000. Adv. Exp. Med. Biol. 477, 349.
      Buhling F., et al. 2000. Adv. Exp. Med. Biol. 477, 241.
      Gerber A., et al. 2000. Adv. Exp. Med. Biol. 477, 287.
      Kas J. et al. 2000. Int. J. Biol. Markers 15, 84.
      Turk B., et al. 2000. Biochim. Biophys. Acta 1477, 98.
      Frosch B.A., et al. 1999. APMIS 107, 28.
      Van Noorden C.J.F., et al. 1997. Anal. Biochem. 252, 71.
      Bossard M.J., 1996. J. Biol. Chem. 271, 12517.
      Traganos F., and Darzynkiewicz, Z. 1994. Methods Cell Biol. 41, 185.
      Guinec N., et al. 1993. Biol. Chem. Hoppe-Seyler 374, 1135.
      Buhling F., et al. Adv. Exp. Med. Biol. 477, 281.
      Leung-Toung R., et al. Med. Chem. 9, 979.
      Product Information
      Detection methodFluorescence
      FormatFluorescence microscopy
      Kit containsLabeled Cathepsin B Substrate, Hoechst 33342 Stain, Acridine Orange, and a user protocol.
      Applications
      Biological Information
      Assay time4-6 h
      Sample TypeCells
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage +2°C to +8°C
      Storage ConditionsUpon arrival store entire kit contents at 4°C. Protect the Cathepsin B fluorogenic substrate from light at all times. Once reconstituted, the 260X Cathepsin B fluorogenic substrate stock should be stored at or below -20°C protected from light. This stock solution is stable for 6 months and should not be thawed more than twice.
      Protect from Light Protect from light
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsLabeled Cathepsin B Substrate, Hoechst 33342 Stain, Acridine Orange, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      219346 0

      Documentation

      References

      Reference overview
      Boonacker E., and Van Noorden, C.J.F. 2001. J. Histochem. Cytochem. 49, 1473.
      Bank U., et al. 2000. Adv. Exp. Med. Biol. 477, 349.
      Buhling F., et al. 2000. Adv. Exp. Med. Biol. 477, 241.
      Gerber A., et al. 2000. Adv. Exp. Med. Biol. 477, 287.
      Kas J. et al. 2000. Int. J. Biol. Markers 15, 84.
      Turk B., et al. 2000. Biochim. Biophys. Acta 1477, 98.
      Frosch B.A., et al. 1999. APMIS 107, 28.
      Van Noorden C.J.F., et al. 1997. Anal. Biochem. 252, 71.
      Bossard M.J., 1996. J. Biol. Chem. 271, 12517.
      Traganos F., and Darzynkiewicz, Z. 1994. Methods Cell Biol. 41, 185.
      Guinec N., et al. 1993. Biol. Chem. Hoppe-Seyler 374, 1135.
      Buhling F., et al. Adv. Exp. Med. Biol. 477, 281.
      Leung-Toung R., et al. Med. Chem. 9, 979.

      Brochure

      Title
      Kit SourceBook - 2nd Edition EURO
      Kits SourceBook - 2nd Edition GBP

      Technical Info

      Title
      Cathepsin B Detection Kit
      User Protocol

      Revision23-August-2010 RFH
      FormatFluorescence microscopy
      Detection methodFluorescence
      StorageUpon arrival store entire kit contents at 4°C. Protect the Cathepsin B fluorogenic substrate from light at all times. Once reconstituted, the 260X Cathepsin B fluorogenic substrate stock should be stored at or below -20°C protected from light. This stock solution is stable for 6 months and should not be thawed more than twice.
      BackgroundElevated cathepsin enzyme activity in serum or the extracellular matrix often signifies a number of gross pathological conditions. Cathepsin-mediated diseases include: Alzheimer's; numerous types of cancer; autoimmune related diseases like arthritis; and the accelerated breakdown of bone structure seen with osteoporosis. Up-regulated cathepsin B and L activity has been linked to several types of cancer. These include cancer of the colon, pancreas, ovaries, breast, lung, and skin (melanoma). Up-regulation of cathepsin K has been shown in lung tumors. Increased cathepsin K activity has also been linked to degenerative bone diseases including osteopetrosis and post-menopausal osteoporosis.

      Cathepsins are usually characterized as members of the lysosomal cysteine protease family and the cathepsin family name has been synonomous with lysosomal proteolytic enzymes. In actuality, the cathepsin family also contains members of the serine protease (cathepsin A,G) and aspartic protease (cathepsin D,E) families as well. These enzymes exist in their processed forms as disulfide-linked heavy and light chain subunits with molecular weights ranging from 20-35 kDa. Cathepsin C is the noted exception, existing as an oligomeric enzyme with a MW ~200 kDa. Initially synthesized as inactive zymogens, they are post-translationally processed into their active configurations after passing through the endoplasmic reticulum and subsequent incorporation into the acidic environment of the lysosomes.
      Principles of the assayThe Calbiochem® Cathepsin B Detection Kit utilizes the fluorophore cresyl violet. When bi-substituted via amide linkage to two cathepsin target sequence peptides {such as (Arginine-Arginine)2}, the cresyl violet leaving group is non-fluorescent. Following enzymatic cleavage at one or both arginine (R) amide linkage sites, the mono and non-substituted cresyl violet fluorophores generate red fluorescence when excited at 550-590 nm.

      The Cathepsin B photostable fluorogenic substrate easily penetrates the cell membrane and the membranes of the internal cellular organelles, enabling researchers to detect cathepsin activity within whole living cells. Intracellular cresyl violet substrate hydrolysis can be monitored by the accumulation of red fluorescent product within various organelles. By varying the duration and concentration of exposure to the Cathepsin B fluorescent substrate, a picture of the relative abundance and intracellular location of cathepsin enzyme activity can be obtained.This activity can be monitored using a fluroescence microscope or plate fluorometer.

      The unsubstituted red fluorescent Cathepsin B product has an optimal excitation and emission wavelength pairing of 592 nm and 628 nm respectively. At these higher excitation wavelengths, the amount of cellular auto-fluorescence is minimal. Furthermore, the excitation peak of this fluorophore is rather broad allowing good excitation efficiency even at 540-560 nm. The typical mercury lamp used in fluorescence microscopy with a maximum light output at 542 nm is therefore quite compatible with the Cathepsin B substrate.

      Hoechst stain is included and can be used to label the cell nuclei after labeling with the Cathepsin B fluorescent reagent. It is revealed under a microscope using an UV-filter with excitation at 365 nm and emission at 480 nm. In addition, acridine orange (AO) is included in the kit to help identify lysosomes and other intracellular organelles. The acidic pH of the lysosome results in the concentration and aggregation of the AO molecules. Aggregated AO molecules fluoresce orange rather than green thus clearly differentiating the lysosomes from the other organelles.
      Materials provided• Cathepsin B fluorogenic substrate (Kit Component No. KP31320) lyophilized
      • Hoechst 33342 stain (Kit Component No. KP31321) 1 ml
      • Acridine orange (AO) (Kit Component No. KP31322) 0.5 ml
      Materials Required but not provided• Cultured cells with media
      • Reagents to induce apoptosis or other experimental condition
      • 15 ml polypropylene centrifuge tube (1 per sample)
      • Amber vials or polypropylene tubes for storage of 260X concentrate aliquots at -20°C
      • Slides and coverslips
      • Hemocytometer
      • Clinical centrifuge at 200 X g
      • 37°C CO2 incubator
      • Pipette(s) capable of dispensing at 10, 50, 200, 300 µl, and 1 ml
      • ddH2O, up to 2 ml needed
      • Phosphate buffered saline (PBS) pH 7.4, up to 100 ml needed
      • Dimethyl sulfoxide (DMSO)
      • Trypsin-EDTA
      • Ice buckets or a refrigerator to maintain cells at 4°C
      • Fluorescence microscope with appropriate filters: excitation 550 nm, emission >610 nm for Cathepsin B fluorogenic substrate; excitation at 480 nm and emission at >540 nm for AO; and if Hoechst is used, a UV-filter with excitation at 365 nm, emission at 480 nm.
      Precautions and recommendations• Use gloves while handling the Cathepsin B fluorogenic substrate, AO, and Hoechst stain.
      • Dispose of all liquid components down the sink and flush with copious amounts of water. • Solid components may be tossed in standard trash bins. A copy of the MSDS is available on-line at www.calbiochem.com.
      PreparationHoechst 33342 Stain: Hoechst stain can be used to label the cell nuclei after labeling with the Cathepsin B fluorogenic substrate. It is revealed under a microscope using a UV-filter with excitation at 365 nm and emission at 480 nm. Hoechst stain is provided ready-to-use at 200 µg/ml.

      Warning: Hoechst stain is a potential mutagen. Use of gloves, protective clothing, and eyewear are strongly recommended. When disposing, flush sink with copious amounts of water.

      Acridine Orange: Acridine orange (AO) is a chelating reagent and can be used to reveal lysosomes, nuclei, and nucleoli. 0.5 ml of AO is provided at 1 mM. AO may be used neat or diluted in ddH2O or media prior to pipetting into the cell suspension. Always protect AO from bright light.

      Lysosomal structures can be visualized using AO concentrations ranging from 0.5 to 5.0 µM. This concentration range can be obtained by diluting the AO reagent stock 1:2000 to 1:200 (0.05-0.5% v/v) into the final cell suspension. For example, if using AO at 1 µM in the final cell suspension, first dilute the AO 1:100 in ddH2O (e.g. put 10 µl AO into 990 µl ddH2O). Then pipette the AO into the cell suspension at 1:10 (e.g. put 55.5 µl diluted AO into 500 µl cell suspension).

      As AO exhibits a very broad emission range, one of several filter pairings on the fluorescence microscope can be used to view this stain. The same excitation/emission filters used to view the Cathepsin B fluorogenic substrate can also be used: a 550 nm (540-560 nm) excitation and long pass >610 nm emission/barrier filter pairing. In this case, the lysosomes will appear red.

      When illuminating with a blue light (480 nm) excitation filter, a green light (540-550 nm) emission/barrier filter combination works well. Lysosomes will appear yellowish green instead of red. As this filter combination is very close to the maximum emission of AO, the slide may appear too bright. Cells read at this combination may need to be washed prior to viewing to remove any excess AO.

      Because of the emissions overlap, dual staining of cells with both Cathepsin B fluorogenic substrate and AO will yield confusing results. Therefore these dyes should not be used to stain the same cells.

      Warning: AO is a potent mutagen and probable carcinogen. Use of gloves, protective clothing, and eyewear are strongly recommended. When disposing, flush sink with copious amounts of water.

      • Reconstitution of the 260X Cathepsin B Fluorogenic Substrate Stock: The Cathepsin B fluorescent reagent is a highly concentrated lyophilized powder. It must first be reconstituted in DMSO, forming a 260X stock concentrate, and then diluted 1:10 in PBS to form a final 26X working solution. For best results, the 26X working solution should be prepared immediately prior to use; however, the reconstituted 260X stock concentrate can be stored in aliquots at or below -20°C for future use. The newly reconstituted 260X Cathepsin B fluorogenic substrate stock must be used or frozen immediately after it is prepared and protected from light during handling.

      1. Reconstitute the vial of lyophilized Cathepsin B fluorogenic substrate with 200 µl DMSO to yield a 260X concentrate.
      2. With the cap on, mix by swirling or tilting the vial, allowing the DMSO to travel around the base of the vial until completely dissolved. At room temperature (RT), the reagent should be dissolved within a few min.
      3. Aliquot and store it at or below -20°C (Section 12).

      If not all of the 260X Cathepsin B fluorogenic substrate stock will be used the same time it is reconstituted, the unused portion may be stored at or below -20°C for 6 months. During that time, the 260X stock may be thawed and used twice. After the second thaw, discard any remaining 260X stock. If you anticipate using it more than twice, make small aliquots in amber vials or polypropylene tubes and store at or below -20°C protected from light. When ready to use, follow Section 13 below.

      • Preparation of 26X Cathepsin B Fluorogenic Substrate Working Solution Just Prior to Use: Using the freshly reconstituted (or thawed) 260X Cathepsin B fluorogenic substrate stock, prepare the 26X working-strength solution by diluting the stock 1:10 in PBS. Mix by inverting or vortexing the vial at RT. Following the suggested protocols here, each 0.5 ml sample to be tested requires only 20 µl of 26X Cathepsin B fluorogenic substrate solution (or 2 µl of the 260X Cathepsin B fluorogenic substrate stock).
      If some of the 260X Cathepsin B fluorogenic substrate was previously reconstituted and then stored at or below -20°C, it may be used 2 more times within 6 months.

      1. Thaw the 260X stock and protect from light.
      2. Once the aliquot has become liquid, dilute the 260X stock solution 1:10 in PBS and vortex. For example, mix 10 µl of 260X stock with 90 µl PBS.
      3. If the 260X Cathepsin B fluorogenic substrate stock was frozen immediately after reconstitution and was never thawed, return it to the freezer. If the stock was thawed once before, discard it.
      4. Proceed to the labeling protocol (Section 14 or 15).

      The 26X working strength Cathepsin B fluorogenic substrate solution must be used the same day that it is prepared.
      Detailed protocol

      Fluorescence Microscopy Staining Protocol for Suspension Cells

      1. Culture cells to a density optimal for the cell line being studied.
      2. Cultivate or concentrate cells to a density of at least 5 X 105 cells/ml. Cells may be concentrated by centrifugation just prior to staining.

      Cell density in the cell culture flasks should not exceed 106 cells/ml. Cells cultivated in excess of this concentration may begin to naturally enter apoptosis due to nutrient deprivation or the accumulation of cell degradation products in the media. Optimal cell concentration will vary depending on the cell line used.

      3. Expose cells to the condition being investigated.
      4. Culture an equal volume of negative control cells in parallel for use as a reference population. Make sure that both the experimental (positive) cell population tubes and the negative control tubes contain similar quantities of cells.
      When ready to label with the 26X Cathepsin B fluorogenic substrate solution, cell density should be between 5 X 105-2 X 106 cells/ml for best viewing.
      5. If necessary, concentrate cells by gentle centrifugation at 200 X g for 3-8 min.
      6. Prepare the 260X stock by reconstituting the vial of lyophilized Cathepsin B fluorogenic substrate with 200 µl DMSO (Section 10).
      7. Prepare the 26X stock by diluting the reconstituted vial with appropriate amount of PBS (Section 11).
      8. Transfer 500 µl of each of the induced and negative control cell suspensions into fresh 12 x 75 mm glass or polypropylene tubes. Or, if desired, larger cell volumes can be used, however more of the 26X solution is required. Larger volume cell suspensions label nicely using 25 cm2 tissue culture flasks (laid flat).
      9. Add 20 µl of the 26X solution directly to each 500 µl cell suspension forming a final volume of 520 µl.
      10. Or, if a different volume was used, add the 26X solution at a 1:26 ratio of the final volume. For example, if 1 ml of cell suspension was used, add 40 µl of the 26X Cathepsin B fluorogenic substrate solution forming a final volume of 1,040 µl. (To optimize this assay to your specific research conditions, adjust the amount of 26X solution used to determine the greatest difference in the fluorescence signal between positive and negative cell populations.)
      Each investigator should titrate the amount of Cathepsin B fluorogenic substrate used to accommodate their particular cell line and research conditions.
      11. Mix the cells thoroughly.
      12. Incubate cells for 15 to 60 min at 37°C under 5% CO2, protecting the tubes from light. As cells may settle on the bottom of the tubes, gently resuspend them by swirling the tubes every 5 min during this incubation time. This will ensure an even distribution of the Cathepsin B fluorogenic substrate among all cells. (To optimize this assay to your specific research conditions, adjust this incubation time to determine the greatest difference in the fluorescence signal between positive and negative cell populations). Each investigator should adjust the incubation time to accommodate their particular cell line and research conditions.
      13. At this point, the cells may be stained with Hoechst stain (Section 8), or AO (Section 9).
      14. If cells are to be monitored using Hoechst stain (Section 8):
      a. Add 2.5 µl Hoechst stain (0.5% v/v) to the 520 µl cell suspension labeled with Cathepsin B fluorogenic substrate.
      b. Or, add 2.5 µl Hoechst stain (0.5% v/v) to a 500 µl cell suspension that was not labeled.
      c. Incubate for an additional 5-10 min at 37°C under 5% CO2.
      d. Go to Step 16.
      15. If cells are to be stained with AO (Section 9) instead of Cathepsin B fluorogenic substrate, pipette the AO reagent stock 1:2000 to 1:200 (0.05-0.5% v/v) into the final cell suspension. (Because of the emissions overlap, dual staining of cells with both Cathepsin B fluorogenic substrate and AO will yield confusing results.)
      a. For example, if using AO at 1 µM in the final cell suspension, first dilute the AO 1:100 in diH2O: put 10 µl AO into 990 µl ddH2O. Then pipette the diluted AO into the cell suspension at 1:10: put 55.5 µl diluted AO into 500 µl cell suspension.
      b. Incubate an additional 30 min at 37°C under 5% CO2
      c. If viewing under the same filters used for the Cathepsin B fluorogenic substrate staining (excitation at 550 nm, emission >610 nm), cells may be viewed immediately after staining, without a wash step - go to Step 16.
      d. If viewing under blue (480 nm) excitation and green (540-550 nm) emission wavelengths, cells may need to be washed with PBS to remove any excess AO as the cells may appear too bright. Brightness will depend on the type of microscope used, and the type of cell line. To wash the cells:
      i) Gently pellet cells at 200 X g for 3-8 min at RT.
      ii) Remove and discard supernatant.
      iii) Resuspend cells in a similar volume of PBS.
      iv) Go to Step 16.
      16. Place 15-20 µl of the cell suspension onto a microscope slide and cover with a coverslip.
      17. Observe Cathepsin B fluorogenic substrate stained cells using a fluorescence microscope equipped with an excitation filter of 550 nm (540-560 nm) and a long pass >610 nm emission/barrier filter pairing. (If these filters are not available, select a filter combination that best approximates these settings.) Using this excitation/emission filter pairing, cells should stain red with more brightly stained vacuoles and lysosomes.
      18. If the same sample was stained with both Cathepsin B fluorescent substrate and Hoechst, and if a multi-wavelength filter option is available on the fluorescence microscope, the dual staining properties of the sample can be examined. Hoechst stain can be seen using a UV-filter with excitation at 365 nm and emission at 480 nm. If these exact filter pairings are not available, select a filter combination that best approximates these settings.
      19. As AO exhibits a very broad emission range, one of several filter pairings on the fluorescence microscope may be used. The same excitation/emission pairing filters used to view the Cathepsin B fluorogenic substrate may be used: a 550 nm (540-560 nm) excitation and long pass >610 nm emission/barrier filter pairing. In this case, the lysosomes appear red. When illuminating with a blue light (480 nm) excitation filter, a green light (540-550 nm) emission/barrier filter combination works well. Lysosomes will appear yellowish green instead of red. Because of the emissions overlap, dual staining of cells with both Cathepsin B fluorogenic substrate and AO will yield confusing results. Therefore, these dyes should be used separately.

      Fluorescence Microscopy Staining Protocol for Adherent Cells

      1. Select the cell culture flask containing the cells that will be studied and dislodge the attached cells.
      a. Aseptically remove the media from the flask.
      b. Depending on the size of your culture flask, aseptically add 5-25 ml sterile PBS or saline to the flask.
      c. Aseptically remove this solution and discard.
      d. Add trypsin reagent to the flask, varying the amount depending on the flask surface area. For example, a 25 cm2 flask should receive ~1 ml of the trypsin reagent; a 75 cm2 flask should receive ~3 ml.
      e. Incubate the flask for 1-2 min, rocking the flask gently back and forth to dislodge the attached cells.
      f. To neutralize the trypsin activity and count the cells, dilute the contents of the flask 1:20 into culture media. For example, take 1 ml of suspension and add to a sterile culture tube containing 19 ml of the complete cell culture media (with serum).
      g. Count the cells using a hemocytometer.
      h. Wash cells by centrifugation at 200 x g for 3-8 min and resuspend cell pellet with fresh medium.
      2. Seed about 104-105 cells onto a sterile coverslip in a 35 mm petri dish or onto chamber slides.
      3. Grow the cells using your culture media formulation until about 80% confluent. This usually takes about 24 h, but will vary with your cell line.
      4. Expose cells to your experimental conditions.
      5. To label cells with the Cathepsin B fluorogenic substrate, simply dilute the 26X substrate solution 1:26 directly into the wells or slide chambers that contain the adherent cells.
      6. For a well or slide chamber that holds 300 µl cell overlay media, add 12 µl of the 26X Cathepsin B fluorogenic substrate solution directly to the cell overlay media to reach a final volume of 312 µl.
      7. Or, if a different volume was used, add the 26X Cathepsin B fluorogenic substrate solution at 1:26. For example, if 1,000 µl of cell media was used, add 40 µl of the 26X Cathepsin B fluorogenic substrate solution forming a final volume of 1,040 µl. (To optimize this assay to your specific research conditions, adjust the amount of 26X Cathepsin B fluorogenic substrate used to determine the greatest difference in the fluorescence signal between positive and negative cell populations.) Each investigator should titrate the amount of Cathepsin B fluorogenic substrate used to accommodate their particular cell line and research conditions.
      8. Gently mix the cell overlay media to ensure even exposure to the Cathepsin B fluorogenic substrate.
      9. Incubate cells for 30-60 min at 37°C in a CO2 incubator.
      10. Remove the media.
      11. Rinse twice with PBS, 1 min per rinse.
      12. At this point, labeled and unlabeled cells can be stained with Hoechst stain (Section 8), and unlabeled cells can be stained with AO (Section 9).
      13. If cells are to be monitored using Hoechst stain (Section 8):
      a. Add 1.6 µl Hoechst stain (0.5% v/v) to each 312 µl of cell overlay material unlabeled or labeled with Cathepsin B fluorogenic substrate.
      b. Incubate for an additional 5-10 min at 37°C under 5% CO2.
      c. Go to Step 15.
      14. If cells are to be monitored using AO (Section 9):
      a. Pipette the AO reagent stock 1:2000 to 1:200 (0.05-0.5% v/v) into the final cell overlay media. Because of the emissions overlap, dual staining of cells with both Cathepsin B fluorogenic substrate and AO will yield confusing results. Therefore, the dyes should be used separately.
      b. For example, if using AO at 1 µM in the final cell media, first dilute the AO 1:100 in diH2O: put 10 µl AO into 990 µl diH2O. Then pipette the diluted AO into the cell overlay media at 1:10: put 111 µl diluted AO into 1000 µl cell overlay media forming a final volume of 1111 µl.
      c. Incubate for an additional 30 min at 37°C under 5% CO2.
      d. Remove the media from the cell mono-layer surface.
      e. Rinse twice with PBS, 1 min per rinse.
      15. Mount the coverslip with cells facing down onto a drop of PBS. If a chamberslide was used, pull off the plastic frame and add a drop of PBS to the cell surface and cover with a coverslip.
      16. Observe Cathepsin B fluorogenic substrate stained cells using a fluorescence microscope equipped with an excitation filter of 550 nm (540-560 nm) and a long pass >610 nm emission/barrier filter pairing. (If these filters are not available, select a filter combination that best approximates these settings.) Using this excitation/emission filter pairing, cells stain red with more brightly stained vacuoles and lysosomes.
      17. If the same sample was stained with both Cathepsin B fluorogenic substrate and Hoechst, and if a multi-wavelength filter option is available on the fluorescence microscope, the dual staining properties of the sample can be examined. Hoechst stain can be seen using a UV-filter with excitation at 365 nm and emission at 480 nm. If these exact filter pairings are not available, select a filter combination that best approximates these settings.
      18. As AO exhibits a very broad emission range, one of several filter pairings on the fluorescence microscope may be used. The same excitation/emission pairing filters used to view the Cathepsin B fluorogenic substrate may also be used: a 550 nm (540-560 nm) excitation and long pass >610 nm emission/barrier filter pairing. Lysosomes will appear red. When illuminating with a blue light (480 nm) excitation filter, a green light (540-550 nm) emission/barrier filter combination works well. In this case, lysosomes will appear yellowish green instead of red. Because of the emissions overlap, dual staining of cells with Cathepsin B fluorogenic substrate and AO will yield confusing results. Therefore, these dyes should be used separately.
      19. To optimize this assay for your specific research conditions, vary the amount of 26X Cathepsin B fluorogenic substrate used (Step 5), and the incubation time (Step 9) to determine the greatest difference in the fluorescence signal between positive and negative cell populations.
      Protocol Summary• Culture cells to the density optimal for your experimental condition, but not to exceed 106 cells/ml.
      • At the same time, culture a non-induced negative control cell population at the same density as the induced population for every labeling condition. For example, if labeling with Cathepsin B fluorogenic substrate, Hoechst stain, and AO, make some or all of these 5 populations:
      a. Unlabeled cells.
      b. Cathepsin B fluorogenic substrate labeled cells.
      c. Cathepsin B fluorogenic substrate and Hoechst labeled cells.
      d. Hoechst labeled cells.
      e. AO labeled cells.
      • Reconstitute the vial of lyophilized Cathepsin B fluorogenic substrate with DMSO to form the 260X stock concentrate. (Section 10).
      • Dilute the 260X stock to the 26X working solution (Section 11 or 13) just prior to use.
      • Stain cells by adding the 26X Cathepsin B fluorogenic substrate working solution.
      • Incubate cells for >1 h.
      • If necessary, wash and spin cells.
      • If desired, label cells with Hoechst stain (Section 8).
      • If desired, label non- Cathepsin B fluorogenic substrate -stained cells with AO (Section 9).
      • Analyze stained cells by fluorescence microscopy.
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