Best to use SMMHC for smooth muscle cells since SMC22 could be expressed by other cells as well

PCR is not really the best way to assess what cells you have in a culture. FACS or immunofluorescence labelling would be better.

If PCR is the only option available then (assuming when you say "SMC22" that you mean transgelin, aka SM22a) it should work fine to detect any smooth muscle cell contamination. You should also consider that if the cells have not been cultured then the fibroblasts (more likely contaminants) will be negative for muscle markers. If the cells have been cultured the fibroblasts will take on a myofibroblast phenotype and are more likely to be positive for SMA/TAGLN.

You have many choices for demonstrating you have urothelial cells here are a couple:

UPK2 - expressed in the most differentiated superficial urothelial cells

KRT7 - highly abundant and expressed in all cell layers of the urothelium

You didn't provide enough information to provide an intelligent answer. First missing piece of information is how were the cells derived, and how have they been cultured? If this is a primary culture, then as Simon comments, fibroblasts are the more likely contaminant. Vimentin staining with IHC or flow cytometry could be very informative. Uroplakin, the definitive marker for urothelials cells, is not expressed in ordinary cell culture.

Dear Robert, thank you very much for your reply! We took out the bladder from mice and did some organ bath experiments lasting up to 6 hours. Then we removed the urothelial cell layer by stripping using foreceps. We do not work with cultured cells. By PCR we now would like to demonstrate the purity of the stripped urothelial layer and the remaining smooth muscle cell layer (no urothelial cells left). Thus, I was looking for selective marker genes. Best ragards Andreas

we have previously used calponin as a marker for separating the mucosa and detrusor muscle. We didn't find Vimentin very useful as it was also found in the myofibroblasts that would be likely to be present in the layer you stripped off, similarly a-smooth muscle actin is found in fibroblasts. We had success with PCR expression of calponin only being present in the detrusor layer and not the mucosal layer.

Dear all, thank you very much for all contributions! Ideed SMMHC was the best marker for the muscle cell layer (exclusion of SMC contamination of the urothelial cell layer preparation) ... calponin was also OK, SM22a was not suitable. Now we will test UPK2 and KRT7 next! Best regards Andreas

Hello Andreas, Any update on your findings? Did the UPK2 and KRT7 work?

If so would you mind sharing the used sequences?

Kind regards

Henk

Hi Henk, both uroplakin-2 and cytokeratin-7 worked perfect als marker for urothelial cells. We used  Qiagen QuantiTect primer assay kits. Good luck!  Andreas

Thank you, will update this section once we get some more info.

I am currently extracting urothelium from intact bladder and I was wondering how you separated the urothelium from the bladder to begin with. I used SMMHC to check for smooth muscle contamination and I found it in my urothelium sample. I was hoping someone could show me their  technique for urithelium separation from fresh intact whole bladder.

it is fairly simple, but there are a lot of steps where it can go wrong. Please look up some articles by Jenny Southgate. Her articles have the best protocols.

Alternatively:

When finding a bladder, make sure that it is as sterile as possible in the lumen.

Place the bladder in large tray in flowcab and open it lengthwise with scissors.  make sure you work as aseptic as possible.

let the urine out and lay the bladder flat with the urotheial sides facing up. 

cut a 2x8cm (or strips which fit into 50ml tube) and place in dissociation medium.

incubate for a couple of hours and you should be able to peel off the urothelial layer using foreceps.

then grab the urothelial layer and scrape the tissue with foreceps and in medium.

collect medium and centrifuge and seed the cells in primaria flasks.

let us know what your final working protocol is.

We also used foreceps to peel of the urothelial layer .... the handling needs some practice!

Using desmin (DES) versus cytokeratin 7 (KRT7) expression works well.  Both transcripts are abundant in smooth muscle and urothelium, respectively.  These transcripts are also not expressed by their counterparts giving a very sensitive assay.

Thank you Henk hogemkamp, my supervisor recently brought me in contact with Jennifer and hopefully I should be able to learn the technique from her personally.