Just try first to measure e.g. beta hexosaminidase activity using coloremetric or fluoremetric substrates...this assay has been published many times.....you can later do the same for amyn other acidic hydrlases.....the cathepsin-B activity assay is very crude and lysosensors are also not very good unless you use ratiometric determinations...

Good luck

You could try the long-lived protein degradation assay. It is generally used to measure flux through the (macro) autophagy pathway, but I imagine it could also be used as a measure of general lysosomal activity. It is an assay that is easiliy applied to any cell type. You readout would be amount of radioactivity in free (degraded) amino acids compared to radioactivity in the proteins of the cell.

Another approach would be to measure EGF-receptor degradation at several time-points after stimulating cells with EGF. This will give you a measure of degradation of the EGF receptor through endocytosis, MVB internalization and processing in lysosomes. The readout here would be through western-blotting for EGF-R.

These two methods look at degradation through alternative pathways so if you have the time and resources, trying both assays would probably give a robust answer in my opinion.

Hi Elisa!

A great researcher that could help you out is Rodrigo Cardoso Oliveira.

Best regards!

I agree with Paul. We work with lysosomal storage diseases and we use beta-hexosaminidase fluorogenic assay to measure enzyme activity by turn over of a labelled substrate as a lysosome marker. Acid phosphotase is more for endosomes not strict lysosomes. Hope this helps.

Thanks for your request, I appreciate your help.

I used long lived protein degradation assay, but my objective now is try to measure only the lysosomal activity, its maduration process and pH, using differents substances... I thought in lysosomal isolation, but is very difficult to do it without guide, because is a "tricky manual".

I think about your tips.

Lysosomal marker? Did you try arylsulfgatase A activity? Use this method: Clin Chim Acta. 1987 Apr 30;164(2):171-80.

A simple chromogenic assay for arylsulfatase A.

Lee-Vaupel M, Conzelmann E.

Abstract

Arylsulfatase A hydrolyzes the artificial chromogenic substrate 4-nitrocatechol-sulfate at 0 degree C at a rate of 24% of that at 37 degrees C whereas arylsulfatase B is almost inactive at 0 degree C. Based on this observation, a simple assay was developed which permits the accurate determination of low residual arylsulfatase A activities in cultured skin fibroblasts of infantile, juvenile and adult MLD patients and pseudodeficient individuals. In cultured skin fibroblasts, the following residual activities were found with this assay system: late-infantile patients, 0.0%, one juvenile patient, 1.0%, adult patients, 4.4-14% of normal average. healthy pseudodeficient probands ranged between 18% and 32%..

Good luck! Klaus Harzer, June 13th, 2014.

The first thing I would like to know, is this: are there lysosomes in your embryonic fibroblasts, they might be very small, and how many/few are they? Have you looked in the microscope after specific staining? I often used (in other material) a sensitive stain for acid PP, for LM, with naphtol-AS-TR-phosphate as substrate. Don't have the reference right now (Barka & Anderson, modified by Lojda). Let me know whether I should search for it.

Thanks for your answer Frank. Yes I have many lysosomes in my MEFs, I labeled with LTR and Lamp1/Lamp2 by IF and with different treatments I checked. I want to verify the pH because I found an alteration in the degradation system combined with a treatment which modifies the internal pH, that's the reason that I want to checked.Anyway I was thinking in alteration in the V-ATPase. Any suggestion? Thank you.

Bafilomycin A1 treatment is the best way to do so. You might find some studies using Con A. Either way both are V-ATPase inhibitors. Just a word of caution - my cells were not happy after leaving BafA1 in for 3 hours. Effect of BafA1 should be visible in 15 minutes.