Microsoft word - ka1330-600310

Background
Lysosomes are single membrane-bound acidic intracellular compartments that break down cellular waste products, fats, carbohydrates, proteins, and other macromolecules into simple compounds. Normal lysosome function enables cells to efficiently mobilize and recycle cellular constituents, but also prevents the accumulation of damaged organelles, misfolded proteins, and invading microorganisms.1 Although lysosomes are found in all animal cells, they are most abundant in disease-fighting cells such as leukocytes. Lysosomes contain about 50 different degradative enzymes, which are optimally active at the acidic pH (about 5) that is maintained within lysosomes but not at the neutral pH (about 7.2) characteristic of the rest of the cytoplasm.2 These enzymes work together to control protein and organelle homeostasis. Disorders of these lysosomal enzymes cause several human diseases including Tay-Sachs disease, arthritis, and neurodegenerative diseases. Evidence shows that in Parkinson’s disease brains, expression of lysosomal enzymes, such as cathepsin D, is significantly decreased. Upregulation of lysosomal genes is beginning to be explored as a potential therapeutic approach against neurodegenerative diseases such as Parkinson’s Disease.3 About This Assay
Lysosome/Cytotoxicity Dual Staining Kit provides a convenient tool for studying lysosome function at the cellular level. The kit employs 4-nitro-7-(1-piperazinyl)-2,1,3-benzoxadiazole (NBD-PZ), which is membrane permeable and reacts with carboxylic acids in the acidic luminal environment of lysosomes,4 as a probe for the detection of lysosomes in cultured cells. Propidium iodide is used as a marker of cell death. Chloroquine, a known inhibitor of lysosome function, is included as a positive control. The kit provides sufficient reagents to effectively treat/stain 960 individual wells of cells when utilized in a 96-well plate format. Lower density plates will still require approximately the same amount of reagents on a per plate basis. Therefore, up to 10 plates worth of cells can be examined irrespective of the number of wells/plate. Exceptions include protocols in which non-adherent cells are utilized and flow cytometry detection methods are employed. Material Supplied
Kit will arrive packaged as a -20°C kit. For best results, remove components and store as stated below. Cell-Based Propidium Iodide Solution 1 vial/250 µl WARNING: This product is for laboratory research use only: not for administration to humans. Not for human or veterinary diagnostic or therapeutic use. Materials Needed But Not Supplied
A 6-, 12-, 24-, or 96-well cell culture plates HepG2 cells (can be obtained from ATCC); other cell lines can also be used Lysosome/Cytotoxicity Dual Staining Kit ( Cat # KA1330 V.01 ) 1 A fluorescence microscope or plate reader capable of detecting NBD-PZ at excitation and emission wavelengths of 485 and 535 nm, respectively, and propidium iodide at excitation and emission wavelengths of 536 and 617 nm, respectively Adjustable pipettes and a repeat pipettor Storage and Stability
This kit will perform as specified if stored as directed in the Materials Supplied section and used before the
expiration date indicated on the outside of the box. Reagent Preparation
Cell-Based Assay Buffer Preparation - Dissolve each Cell-Based Assay Buffer Tablet in 100 ml of distilled water. This buffer should be stable for approximately one year at room temperature. NBD-PZ/Propidium Iodide Dual Staining Solution Preparation - To 10 ml of culture medium used for your cells, add 10 µl of NBD-PZ Stock Solution and 20 µl of Cell-Based Propidium Iodide Solution (Catalog No. 10011234). Mix well. Protect from light. • NBD-PZ is extremely light sensitive and is photobleached quickly. All staining procedures must be performed without direct exposure to intense light. Therefore, incubations need to be done in the dark. • For all assay protocols, it is imperative that samples be analyzed immediately following completion of the staining. It is recommended that NBDPZ staining be measured first and the propidium iodide staining be measured second to avoid depletion of the NBD-PZ fluorescence during analysis. Performing the Assay
The following protocol is for 96-well plates. The volume of buffer and staining solution should be adjusted accordingly if you are using a different plate density. Seed a 96-well plate with 5 x 104 cells/well. Grow cells overnight. The next day, treat the cells with experimental compounds or vehicle control for 24-72 hours, or for the period of time used in your typical experimental protocol. To use the Chloroquine Positive Control, dilute 1:1,000-1:5,000 into your culture medium. After the treatment period, centrifuge the plate for five minutes at 400 x g at room temperature. Add 100 µl of the NBD-PZ/Propidium Iodide Dual Staining Solution to each well. Be careful to not disturb the cell layer. Incubate the cells for 10 minutes in a cell culture incubator at 37°C. Centrifuge the plate for five minutes at 400 x g at room temperature. Add 100 µl of Cell-Based Assay Buffer to each well. Be careful to not disturb the cell layer. The cells are now ready for analysis by fluorescent microscopy and must be analyzed immediately. Lysosome/Cytotoxicity Dual Staining Kit ( Cat # KA1330 V.01 ) 2 Lysosomes stained by NBD-PZ can be detected with a filter capable of excitation and emission at 485 nm and 535 nm, respectively. Dead cells are stained by propidium iodide and can be detected with filters usually designed to detect rhodamine (excitation/emission = 540/570 nm, respectively) or Texas Red (excitation/ emission = 590/610 nm, respectively). The following protocol is designed for a 96-well plate. Adjust volumes accordingly for other plate densities. A 96-well black culture plate should be used for this method. Optimal conditions will depend on the cell type being Seed a 96-well black culture plate with 5 x 104 cells/well. Grow cells overnight. The next day, treat the cells with experimental compounds or vehicle control for 24-72 hours, or for the period of time used in your typical experimental protocol. To use the Chloroquine Positive Control, dilute 1:1,000-1:5,000 into your culture medium. NOTE: Differences in cell density can significantly affect results. Ensure that experimental compounds do not significantly inhibit cell proliferation. After the treatment period, centrifuge the plate for five minutes at 400 x g at room temperature. Add 100 µl of the NBD-PZ/Propidium Iodide Dual Staining Solution to each well. Be careful to not disturb the cell layer. Incubate the cells for 10 minutes in a cell culture incubator at 37°C. Centrifuge the plate for five minutes at 400 x g at room temperature. Add 100 µl of Cell-Based Assay Buffer to each well. Be careful to not disturb the cell layer. The cells are now ready for analysis in a plate reader and must be analyzed immediately. Lysosome staining intensity can be detected by using an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The degree of cell death can be assessed by measuring propidium iodide staining intensity at excitation and emission wavelengths of 536 and 617 nm, respectively. Seed cells in a 6-, 12-, or 24-well plate at a density of 105-106 cells/well in 2, 1, or 0.5 ml of culture The next day, treat the cells with experimental compounds or vehicle control for 24-72 hours, or for the period of time used in your typical experimental protocol. To use the Chloroquine Positive Control, dilute 1:500-1:10,000 into your culture medium. At the end of the treatment, trypsinize (adherent cells) or collect cells (suspension cells). Centrifuge at 400 x g for five minutes to pellet the cells. Resuspend cell pellet in 0.5 to 1 ml of NBD-PZ/Propidium Iodide Dual Staining Solution. It is important to achieve a monodisperse cell suspension at this step by pipetting up and down repeatedly. Incubate the cells for 10 minutes in a cell culture incubator at 37°C. Lysosome/Cytotoxicity Dual Staining Kit ( Cat # KA1330 V.01 ) 3 Centrifuge at 400 x g for five minutes to pellet the cells. Resuspend cell pellet in 0.5-1.0 ml of Assay Buffer depending on cell number. Pipette up and down repeatedly to achieve a monodisperse cell suspension. The cells are now ready for analysis by a flow cytometer and must be anyalyzed immediately. Lysosomes staining can be detected in the FL1 channel and dead cells stained by propidium iodide can be detected Cell Staining
Figure 1. Chloroquine increases lysosome accumulation but not cell death in HepG2 cells as measured by fluorescent microscopy. HepG2 cells were seeded at a density of 5 x 104 cells/well and incubated overnight at 37°C. The next day, cells were treated with either vehicle or different concentrations of chloroquine. On the third day, cells were processed for NBD-PZ and propidium iodide staining according to the protocol described above. Panel A: HepG2 cells treated with vehicle. There was a basal level of lysosome staining, indicated by faint green staining of NBD-PZ. Few dead cells were detected (red nuclei staining by propidium iodide). Panel B: NBD-PZ and propidium iodide staining of HepG2 cells treated with 12.5 µM chloroquine. Note the increase in NBD-PZ fluorescence intensity but not the numbers of propidium iodide positive dead cells compared to the Lysosome/Cytotoxicity Dual Staining Kit ( Cat # KA1330 V.01 ) 4 Plate Reader Fluorescence Detection
Figure 2. Chloroquine increases lysosome accumulation but not cell death in HepG2 cells. HepG2 cells were seeded in a 96-well plate at a density of 5 x 104 cells/well and incubated overnight at 37°C. The next day, cells were treated with different concentrations of chloroquine overnight. On the third day, cells were stained with NBD -PZ/Propidium Iodide Dual Staining Solution according to the protocol described above. Left panel: Chloroquine treatment increased NBD-PZ (lysosome staining) fluorescence intensity, indicating that chloroquine caused an increase in lysosome accumulation in HepG2 cells. Right panel: Chloroquine treatment did not cause an increase in propidium iodide staining, indicating that at the concentrations used here, chloroquine did not cause cytotoxicity in HepG2 cells. Flow Cytometry
Figure 3. Chloroquine increases lysosome accumulation and cytotoxicity in Jurkat cells. Jurkat cells were seeded in a 6-well plate at a density of 5 x 105 cells/well in RPMI culture medium and incubated overnight at 37°C. The next day, cells were treated with different concentrations of chloroquine overnight. On the third day, cells were stained with NBD-PZ/Propidium Iodide Dual Staining Solution according to the protocol described above. Left panel: Cells treated with vehicle showed a low percentage of cell death (4%-upper left and upper right quadrants) and about 11% of cells had detectable lysosome staining (lower right and upper right quadrants). Middle panel: When cells were treated with 12.5 µM chloroquine there was not an increase in the percentage of cell death (4.5%-upper left and upper right quadrants) but there was an increase in detectable lysosome staining (24.9%-lower right and upper right quadrants). Right panel: 50 µM chloroquine treatment caused a significant increase in both cell death (8.7%-upper left and upper right quadrants) and lysosome accumulation (36%-lower right and upper right quadrants). Lysosome/Cytotoxicity Dual Staining Kit ( Cat # KA1330 V.01 ) 5 Troubleshooting
High level of propidium iodide Cells are not healthy or are dead Use only healthy living cells References
Fehrenbacher, N. and Jäättelä, M. Lysosomes as targets for cancer therapy. Cancer Res. 65(8), Zhang, L., Sheng, R., and Qin, Z. The lysosome and neurodegenerative diseases. Acta Biochim. Biophys. Sin. 41(6), 437-445 (2009). Schneider, L. and Zhang, J. Lysosomal function in macromolecular homeostasis and bioenergetics in Parkinson’s disease. Mol. Neurodegener. 5, 14 (2010). Ishiguro, K., Ando, T., and Goto, H. Novel application of 4-nitro-7-(1-piperazinyl)-2,1,3-benzoxadiazole to visualize lysosomes in live cells. Biotechniques 45(4), 465-468 (2008). Lysosome/Cytotoxicity Dual Staining Kit ( Cat # KA1330 V.01 ) 6

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