Ab253391 Lysolivetm Lysosomal Β-Galactosidase Assay Kit

Version 1 Last updated 11 August 2020

ab253391 LysoLiveTM Lysosomal β-Galactosidase Assay Kit

View LysoLiveTM Lysosomal β-Galactosidase Assay Kit datasheet: www.abcam.com/ab253391 (use www.abcam.cn/ab253391 for China, or www.abcam.co.jp/ab253391 for Japan)

For monitoring beta-galactosidase activity in lysosomes in live cells.

This product is for research use only and is not intended for diagnostic use.

1. Overview 3 2. Materials Supplied and Storage 4 3. Materials Required, Not Supplied 4 4. General guidelines, precautions, and troubleshooting 5 5. Assay Procedure 6 Technical Support 8

Lysosomes are acidic cytoplasmic organelles that are present in all nucleated mammalian cells. Lysosomes have been found to be involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis, and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of neurological diseases including Parkinson’s, Tay-Sachs, Sandhoff, Krabbe, Wolman, and Gaucher syndromes.

Krabbe Disease is an autosomal recessive disorder that results from a deficiency in an enzyme known as galactocerebrosidase (galactosylceramidase, GALC). Defects in the gene coding for lysosomal galactocerebrosidase result in this lysosomal storage disease, affecting about 1 in 100000 live births. Krabbe Disease is also called Globoid Cell Leukodystrophy. This name derives from the characteristic pathology of Krabbe Disease, where macrophages accumulate high levels of undegraded galactolipids as a result of the lack of GALC activity. These cells produce a characteristic morphology difference from healthy cells, and are often termed globoid cells.

We have developed unique targeted lysosomal staining compounds that are useful for labeling lysosomes in a live-cell format and are capable of simultaneously monitoring lysosomal metabolic activity. These new targeted substrates are based upon fluorescent probes that have a low pKa value for optimal fluorescence at the lower physiological pH values found in the lysosomes as well as specific targeting groups to direct their accumulation to the lysosomes using a live-cell staining format.

The LysoLiveTM Lysosomal β-Galactosidase Assay Kit utilizes a sensitive substrate for lysosomal beta-galactosidase, an enzyme indicated in several diseases such as Krabbe disease and Morquio Syndrome. The substrate only becomes fluorescent upon cleavage by the enzyme meaning lysosomes with low beta-Galactosidase activity will have reduced staining in a manner proportional to the enzyme activity levels found in the cell.

Store kit immediately on receipt. Kit can be stored for 6 months from receipt, if components have not been diluted. Fluorescent reagents should be handled with care, kept cold when not in use, and stored frozen (-20°C). High background fluorescence readings for blank samples will indicate decomposition of the dyes.

Note: Individual units of LysoLiveTM GalGreen may be centrifuged briefly before use to settle contents to bottom of the vial. Partial evaporation of the solvent may have occurred. This does not affect the amount of GalGreen in the vial. When diluting with media, we recommend rinsing the vial repeatedly to ensure all the reagent has been removed.

Aliquot components in working volumes before storing at the recommended temperature.

Avoid multiple freeze thaws.

Storage Item Quantity temperature

LysoLiveTM GalGreen 5 x 100 µL -20°C

Opti-Klear™ Live Cell 2 x 30 mL 4°C Imaging Buffer 1X

3. Materials Required, Not Supplied

These materials are not included in the kit, but will be required to successfully perform this assay: - Tissue culture media

- Sterile distilled H2O - 1X PBS

Please observe safe laboratory practice and consult the safety datasheet. For general guidelines, precautions, limitations on the use of our assay kits and general assay troubleshooting tips, particularly for first time users, please consult our guide: www.abcam.com/assaykitguidelines For typical data produced using the assay, please see the assay kit datasheet on our website.

NOTE: All reagent preparation and cell staining procedures should be performed under sterile conditions, such as in a laminar flow hood.

5.1 If a biological agent is being tested for its effects on enzyme activity, treat cells in culture prior to use of this kit. 5.2 Remove growth medium from cells by aspiration. 5.3 Wash cells with 1X Phosphate Buffered Saline (PBS). Remove by aspiration. 5.4 Prepare staining medium by diluting LysoLiveTM GalGreen 1:1000 in growth medium. 5.5 Add the staining medium from Step 5.4 to cells. See Table 1 for recommended volumes for different culture vessels. 5.6 Incubate cells at 37oC/5% CO2 for 2-16 hours. Inspect the cells by fluorescence microscopy for staining during this interval to determine the optimum staining time(s) for your particular cell type. 5.7 Aspirate staining medium. Wash the cells 3 times with 1X PBS. 5.8 Add fresh growth medium to cells. Alternatively, replace the media with Opti-Klear™ Live Cell Imaging Buffer 1X that can help lower background and improve signal to noise. 5.9 View cells using a fluorescence microscope system equipped with bandpass filters capable of visualizing the green fluorescence channel, i.e. with EX/EM: 490/520 (such as Nikon B-2A, Omega Optical XF14-2, Omega Optical XF68).

NOTE (1): It is recommended that the LysoLiveTM GalGreen be diluted in serum-free growth medium appropriate for the cell line to be assayed. Use of serum may produce inaccurate results due to potential exogenous enzyme activities as well as other components present in serum. It is also suggested that medium used for staining be free of antibiotics/antimycotics, to avoid any potential effects of these compounds on enzyme activities within the cells.

NOTE (2): It is strongly recommended that users determine their own optimum staining concentrations and incubation times. Optimum conditions can vary greatly depending on cell line, culture conditions, and sensitivity of fluorescence microscopy equipment.

Copyright © 2020 Abcam. All rights reserved Rec. Vol. Staining Culture Vessel Medium Rec. Vol. 1X PBS 100MM CULTURE DISH 10 ML 5 ML 60MM CULTURE DISH 4 ML 2 ML 6-WELL CULTURE PLATE 2 ML/WELL 1 ML 12-WELL CULTURE PLATE 1 ML/WELL 0.5 ML 24-WELL CULTURE PLATE 0.5 ML/WELL 0.25 ML 96-WELL CULTURE PLATE 100 μL/WELL 100 μL Table 1. Recommended reagent volumes for different culture vessels.

Figure 1: Labeling cells with LysoLiveTM Lysosomal β-Galactosidase Assay Kit 15uM LysoLiveTM GalGreen, 16 hour incubation

Left panel: healthy cells, GM14643 Right panel: cells from a Krabbe Disease patient, GM06805

For all technical or commercial enquiries please go to: www.abcam.com/contactus www.abcam.cn/contactus (China) www.abcam.co.jp/contactus (Japan)