If you received exactly the same values (i.e. to the same decimal point) from 2, 3 and 5 ug samples, it suggests that the fault lies in the setup of the microplate reader rather than your assay. If you can see a visible difference in the samples, it could be that the signal intensity of your sample is above the read limit of the plate reader, in which case you would just adjust the sensitivity of the plate reader.

The problem is that I can see a difference in the samples in the first step but not in the last one. I've tried twice (following exaclty the protocol in attach to the kit) with the same bad results.

OK, so according to Fig 1 of the manual, (A), (B) and (C) look right in your experiment, but (D) does not? Also, what does the 1ug sample look like?

I've obtained a value of 0,9 for 2,3 and 5 ug and 0,6 for 1 ug (completely differerent from how reported in the manual). Moreover, while I saw color differences especially between the 1,2 and 3 ug microtube after adding the Dye Reagent, this differences were no longer detectable after adding the dissociation reagent.

If you still see a pellet after you drain the eppendorf (C) and then there is no colour change after you add the dissociation reagent and completely dissolve the pellet, the dissociation reagent could be gone off I guess? Try fresh reagents prehaps.

However, since you are getting the same reading for all three samples in the spectrometer, it could be your spectrometer setup in not sensitive enough to pick up the differences (the "values" you get from your spectrometer don't have to match the kit as the spectrometers can be different). 620nm is not optimal so that could be a problem. Sorry I can't be of more help and best of luck finding a solution.

All the reagent are new (I opened them yesterday). I can see the pellets after the centrifugation (and they seems also different among them). Tomorrow I'll try again. Thanks anyway for the advices!

Just to update my results, I've used concentrations of reference standard solution between 0,1 ug and 1,5 ug and everything works well. I can't explain why it doesn't work with the concentrations suggested in the manual of the kit.

Great stuff, glad you got it working in the end, it looks like the standard was gone off somehow. Well done anyway :)

We used chondroitin sulphate (CS) and its ability to bind alcian blue to set up a standard curve for GAGs. What molecule is used for Standard curve in the Blyscan Kit ? The DMMB assay is another dye binding (spectrophotometric) assay for GAGs. We also found that the DMMB assay only works at a certain range of concentrations of CS. So, there is some tricky stoichiometry regarding no. of dye molecuels binding a certain no. of GAGs to form the complex. I hope this helps!

Also in the Blyscan Kit there is chondroitin sulfate as standard. I hope we can figure it out at the end!! thanks for the help!

The most critical step in the sGAG assay(BLYSCAN) is the draining of the eppendorf tubes carefully not allowing any of the deposit to flow off.My values generally range as follows:

0 ug-0.06

1ug-0.3

2ug-0.6

3ug-0.8

4ug-1.08

5ug-1.3

My correlation coefficient(R sq. value) is usually 0.98-0.99.

Hope this helps.

Charan, have you established reproduciblity andr ange of linearity for the BLYSCAN assay? Can you take a piece of cartilage (complexsGAG) or enzyme digested cartilage and get reliable readings with this assay ? We tried the DMMB (dimethymethylene blue) assay with Synovial fluid. Even with serial dilutions of the cntrifuged S.fluid, we got weird readings. So...wondered what you can do with the BLYSCAN asay.

Hi Dr.Venil.I have established good reproducibility with mesenchymal stem cells differentiated to chondrocytes.I get pretty close readings.I have differentiated about 15 lots of MSCs to chondrogenic lineage and then have performed the BLYSCAN Assay.The key here is normalize the sGAG content to the DNA and then you will see consistency.The BLYSCAN kit also uses the DMMB.

The kit comprises of the extracted sGAG standard from which you can establish a standard curve.

Hope this helped you.Regards.

Interesting Charan, The DMMB assay did work well for monitoring release of sGAGs from explants of OA (osteoarthritic)cartilage. I am glad it works for your model. Truly differentied hyaline cartilage has zones of GAGs with different levels of sulphation. I recall Saffranin O stain being used to show this. Can your MSCs go all the way and becoming mature cartilage?? perhaps Saffranin O stain may help you.

Dr.Venil,We do a reference staining with alcian blue which is meant to stain glycosaminoglycans.When i do this staining on monolayer culture for differentiated MSCs i also see that it stains as patches or zones,now i can relate to what u meant by saying zones of GAGs with different levels of sulphation.

Saffranin O is also widely used in the differentiation model all over the world.But it seems that saffranin O has an affinity to stain chondroitin sulphate,thats the reason we chose BYSCAN(DMMB) that it stains all the sulphated GAGs.

Charan, you may want to quantitate the zones of alcian blue or Saf.O stain. Why? Because the levels of sulfation in mature cartilage are required for its load bearing property..and if you can show your differentiated MSCs can handle load, and correlate it with the gradient of Saf.O stain.--will be a great structure-function assay!

Absolutely!We are currently working with a few biomaterial experts from IIT Delhi to construct silk based scaffold for testing the functionality of cartilage repair.I am really looking forward for the scaffolds.What do you think Dr.Venil?

Do you have better ideas to test cartilage function?

There are some shear-stress studies done under special in vitro conditions. I did not get details but you can search and find them. The different collagen isoforms also vary (I think?) in the sGAG matrix as hyaline cartilage matures. As a neg. control, you could take fibrillar (non-hyaline) cartilage --stuff that comes up after wound healing, and check out all the parameters in parallel--may establish some interesting marker profiles. Will add More if I get ideas.

We did not use the blyscan kit. We bought Sigma's chondrotin sulphate (CS) and dissolved it in dd H2O to make a stock. ALiquots were kept at -20deg C. After thawing, we kept the aliquot of CS at 4deg c, and reused it for 3-4 assays. The DMMB dye was freshly prepared each time (as per SIgma Chemincals instructions). Charan can comment also. Hope this helps!

Yes, diluted working stock of CS was also made in dd H2O. The standard curve can be tried a few times separately-to make sure you get a good straight line. Once it is working, you can do 3 concnetrations (low, med and high), and run unknown samples. You can check out our papers in J.Biosci 2007 for more details..These are free to download. Hope this helps!

Dear Neha,

Yes the CS standards are made with dd H2O in the BLYSCAN kit.It is actually 1 ml of DMMB per 100 ul of test sample that i usually use.It completely depends on the test item you are looking for.You can vary the volume of your test to an extent but i suggest do not alter the 1ml DMMB.

The dissociation reagent to be used is 0.5 ml and not 1ml.Should be run in duplicate in a 96 well plate using 200 ul each of the standard and test.

Yes i suggest you that you run the standard at the same assay/same time of the test estimation.

BLYSCAN is a good kit.You can also follow the procedure in Dr.Venils papers as those are also well established and reproducible.

Best regards

Charan

Hello charan! Hope you are doing well. I had a couple of questions too regarding the Blyscan kit. How long do you digest the monolayer culture with papain? And are you using water as reagent blank? How are you measuring the DNA?

Hi Mam,

Nice to hear from you. I'm doing good. I digest the monolayer culture for 3-3.5 hours at 65 degrees with papain. Yes I use water as my reagent blank.

I extract the DNA with the regular qiagen kit and then estimate it using the Quant IT picogreen DNA estimation kit.This method works the best for the normalization of the sGAG with the DNA content.I have got very consistent results with over 15 batches of the IMP.

With best regards

Charan

Just to clarify regarding the reagent blanks.I use 100ul of water and process the blank just the way i process the other standards i.e add the DMMB,incubate for 30 mins on a shaker then spin,removal of the sup and then addition of the dissociation solution(0.5ml).

regards

Charan

Thank you Charan. So you isolate DNA from a parallel dish of differentiated cells? can it not be quantified using UV spec at 260 nm?

Yes we isolate DNA from a parallel dish(In duplicates for both sGAG and DNA).I suppose it can be quantified using 260nm UV.The pico green is a lot more accurate.

Here is the attached Quant IT reference sheet.

Thank you so much. Can the sGAG assay be stopped at any point in between? After the papain digestion, can the sample be stored?

Useful info Charan ! Why is papain preferred to trypsin?

Dear Venil,

I have no other reason to think of except of the capability of papain activity at 65 degrees.I think trypsin or collagenase would degrade easily at this temperature.Maybe the proteoglycan extraction is best suited at 65 degrees with papain.

The papain digest can be stored at -80.I have assayed samples after a month for repeat analysis,It works.

Yes, Charan, I believe the higher temp tolerence for papain is the main reason. In addition to papin, foods rich in related enzymes (like bromelain from pineapple) are supposed to be good for "healthy joints". Maybe we need some chronic active enzyme to keep joints flexible?! Thanks.