Magic Red® Cathepsin L Assay Kit
Quantitate and monitor intracellular cathepsin L activity over time in vitro. The Magic Red reagent in this assay fluoresces red upon cleavage by active cathepsin enzymes. Analyze the fluorescent signal using fluorescence microscopy or a fluorescent plate reader.
- Prepare samples and controls
- Reconstitute Magic Red by adding 50 µL DMSO.
- Dilute Magic Red 1:10 by adding 450 µL diH2O.
- Add 20 µL Magic Red to each sample (~ 500 µL aliquot of cultured cells).
- Incubate while protected from light.
- Watch color start to develop within 15 minutes.
- If desired, label with additional stains, such as Hoechst, DAPI, Acridine orange, or an antibody.
- If desired, fix or embed cells.
- Analyze with a fluorescence microscope or fluorescence plate reader. Magic Red has an optimal excitation at 592 nm and emission at 628 nm.
If working with adherent cells, please see the manual for additional protocols.
Lo, C;O’Connor, L;Loi, G;Saipuljumri, E;Indajang, J;Lopes, K;Shirihai, O;Grinstaff, M;Zeng, J. Acidic nanoparticles restore lysosomal acidification and rescue metabolic dysfunction in pancreatic β-cells under lipotoxic condition. bioRxiv. 2023 July
Ezz, MA;Takahashi, M;Rivera, RM;Balboula, AZ. Cathepsin L regulates oocyte meiosis and preimplantation embryo development. Cell proliferation. 2023 July
Zhang, C;Chen, H;Sun, L;Zhao, P;Qi, C;Yang, Y;Si, A;Qian, Y;Jung, YS. Bis-Benzylisoquinoline Alkaloids Inhibit Porcine Epidemic Diarrhea Virus by Disrupting Virus Entry. Pathogens (Basel, Switzerland). 2023 June
Zeng, J;Acin-Perez, R;Assali, EA;Martin, A;Brownstein, AJ;Petcherski, A;Fernández-Del-Rio, L;Xiao, R;Lo, CH;Shum, M;Liesa, M;Han, X;Shirihai, OS;Grinstaff, MW. Restoration of lysosomal acidification rescues autophagy and metabolic dysfunction in non-alcoholic fatty liver disease. Nature communications. 2023 May
Rodrigues, PM;Sousa, LG;Perrod, C;Maceiras, AR;Ferreirinha, P;Pombinho, R;Romera-Cárdenas, G;Gomez-Lazaro, M;Senkara, M;Pistolic, J;Cabanes, D;Klein, L;Saftig, P;Alves, NL. LAMP2 regulates autophagy in the thymic epithelium and thymic stroma-dependent CD4 T cell development. Autophagy. 2022 May 19; doi: 10.1080/15548627.2022.2074105. Full Text
Yang, Y;He, X;Xia, S;Liu, F;Luo, L. Porphyromonas gingivalis facilitated the foam cell formation via lysosomal integral membrane protein 2 (LIMP2). Journal of Periodontal Research. 2020 Dec 29; doi: 10.1111/jre.12812. Article
Sun X, Shu Y, Yan P, Huang H, Gao R, Xu M, Lu L, Tian J, Huang D, Zhang J. Transcriptome profiling analysis reveals that ATP6V0E2 is involved in the lysosomal activation by anlotinib. Cell Death Dis. 2020 Aug 24;11(8):702. doi: 10.1038/s41419-020-02904-0. Full Text
He M, Zhang T, Zhu Z, Qin S, Wang H, Zhao L, Zhang X, Hu J, Wen J, Cai H, Xin Q, Guo Q, Lin L, Zhou B, Zhang H, Xia G, Wang C. LSD1 contributes to programmed oocyte death by regulating the transcription of autophagy adaptor SQSTM1/p62. Aging Cell. 2020 Mar;19(3):e13102. doi: 10.1111/acel.13102. Epub 2020 Feb 19. Full Text
Lu K, Zimmermann M, Görg B, Bidmon HJ, Biermann B, Klöcker N, Häussinger D, Reichert AS. Hepatic encephalopathy is linked to alterations of autophagic flux in astrocytes. EBioMedicine. 2019 Oct;48:539-553. doi: 10.1016/j.ebiom.2019.09.058. Epub 2019 Oct 21. Full Text
Matsumoto A, Pasut A, Matsumoto M, Yamashita R, Fung J, Monteleone E, Saghatelian A, Nakayama KI, Clohessy JG, Pandolfi PP. mTORC1 and muscle regeneration are regulated by the LINC00961-encoded SPAR polypeptide. Nature. 541(7636): 228-232. Abstract.
Marwaha R, Arya SB, Jagga D, Kaur H, Tuli A, Sharma M. The Rab7 effector PLEKHM1 binds ArI8b to promote cargo traffic to lysosomes. J. Cell. Biol. 2017. 216(4): 1051-1070. Full text.
Kang HT, Park JT, Choi K, Kim Y, Choi HJC, Jung CW, Lee YS, Chul Park S. Chemical screening identifies ATM as a target for alleviating senescence. Nat Chem Biol. 2017. Jun;13(6):616-623. Epub 2017 Mar 27. Abstract.
Guo C, Zhu Z, Guo Y, Wang X, Yu P, Xiao S, Chen Y, Cao Y, and Liu X. Heparanase upregulation contributes to porcine reproductive and respiratory syndrome virus release. J. Virol. 2017. May 10. pii: JVI.00625-17. doi: 10.1128/JVI.00625-17. [Epub ahead of print]. Abstract.
Question: When using a fluorescence plate reader staining for adherent cell, do I have to detach the cell by trypsin treatment? In that case, Is there anything to be aware of?
Answer: If you are using adherent cells, you do not necessarily need to detach them. If your plate reader is capable of reading from the bottom, you can grow your adherent culture in a tissue culture plate with black walls and a clear bottom.