Refer these protocols.

http://www2.cbm.uam.es/mkfactory.esdomain/webs/cbmso/images/TextBox/calcio.pdf

http://physrev.physiology.org/content/79/4/1089.long

incubate the lymphocytes for 1 hr on poly-l-lysine and use within 5 hrs. Smooth muscle should attach overnight.

For the lymphocytes, if you do not need to image individual cells, you could measure [Ca2+] in a population in suspension. For this, you could use a stirred cuvette in a microfluorimeter, a fluorescent plate reader, or Fluorescence Activated Cell Sorting (FACS).

I am not too sure about the problem with smooth muscle adherence. I use a smooth muscle derived cell-line (A7r5) that adheres directly onto glass coverslips. For your cells, maybe you could use a different type of coating, such as collagen?

I am working with bone marrow-derived mast cells so I don´t have experience with the cells you use. But in my case centrifuging the cells and re-suspending them in fresh medium makes them attach much better to PLL. However, I also measure within 1 hour of plating.

I have also noticed that cells in bad shape don´t attach as good as healthy cells from a good culture.

I had the same problem with the aortic smooth muscle cells. Now, I am using Matrigel from BDBioscience with the dilution of 1:30-1:50. The Matrigel creates a semi ECM environment for you cells. I am using the one with reduced growth factor. It is easy to use and the coating takes only 20-30 minutes. Although I prefer to coat my dishes almost 1 hour before seeding my cells. Hope this is helpful. Good Luck.

Thanks for your contribution.

There is another problem, though the lymphocytes in culture look healthy and nice in shape, sometime they don´t respond to Carbachol or Achetylcholine. Do you have any explanation??

I have another idea for cell attachment, peptide hydrogel from BD bioscience.

For muscle cells, people using poly-ornithine.

lymphocytes don't respond to Cch or Ach; no muscarinic receptors. Try anti-CD3 antibodies or PHA.

@ Jonathan: Thanks.

Actually in the beginning of my experiment I was thinking there is muscarinic receptor in lymphocyte. Also I found couple of articles though those haven´t made any concrete conclusion about presence of muscarinic receptor but they have shown expression of muscarinic mRNA in T-lymphocytes.

Could you please give one reference article?

Ok

Article STIM1 is required for attenuation of PMCA-mediated Ca2+ clea...

Hi, Thanks. Very helpful information.

Why do you used 11 mM Glucose? Isn´t it stimulatory dose? I am using Ca-5 buffer which contain 3.3 mM glucose.

Another thing, I have used CCh or Ach because they go through IP3. So I am trying to find out if there anything I could use as positive control which will go through IP3 receptor.

Thanks again for your nice paper.

The buffer is standard for this Calcium experiments; I have some some version of it for 20 years and recommend it to you. If you are using T lymphocytes, the approaches used in my paper will work. The T Cell Receptor is PLC-coupled; PLC is the enzyme that produces IP3 - many different GPCRs and TKRs do this.

Hi, Here is one nice article...

Hi Muhammad, it is unusual that the SMC do not adhere, usually they should see within minutes glass or plastic no treatment - without being patronising what are your steps prior to seeding - question is, are you damaging the cells in the harvest such as too harsh centrifugation, too long in trypsin.

Hi, I did one experiment where I found very small number of cells. But after then I could not see any cells. I found one problem with incubator.

But I can explain my protocol.

I used 0.25% , I ml trypsine for 75 CM2 culture flask. After adding trypsine 4 min incubation inside the incubator, then I took off the trypsin and put the flask back inside the incubator for another 3 min, then added culture medium. Then collect the cell along with culture medium and centrifuge in 200 G for 5 min.

I am not sure of the removal of the trypsin stage, you could be loosing a lot of cells then. Also time in trypsin is long. Centrifugation speed seems fine, the cell pellet should be loose enough that adding fresh media displaces it without much agitation.

Here is how I would do it:

- remove media from flask

- wash cells in flask x2 in warm PBS (-Ca, -mg)- to remove all calcium from media that can inactivate the trypsin. (remove all excess PBS

- add trypsin (1ml of 10x, not diluted) and incubate for about 1-2 minutes in incubator and check under scope for detachment. SMC should come off easily like this and it reduces the time in trypsin. I have tried diluted trypsin but it takes longer and the cells don't fare as well.

- I would then immediately stop trypsin buy adding fresh media (9ml, don't remove the trypsin) and remove the contents of the flask to a sterile tube i.e. 15ml

- I would count cells quickly, as SMC settle and adhere quickly

- then I would then gently agitate (invert once) the tube containing cells, to ensure equal mixing of cells (the settle quick) and seed them directly at the required number onto your glass coverslips e.g. 5x103-1x104 in 200ul in a bubble on the coverslip

- The cells will settle and adhere very quickly, you should be able to look at them (carefully) after 2 hours and see an almost confluent monlayer. Then add 1ml media to the well and let settle overnight.

Hopefully this will help, let us know!

Scott

Thanks Scott Johnstone. Do you have any standard protocol for human colonic smooth muscle cell isolation from tissue?

In brief, I am using, 2 mg/ml collagenase, in RPMI with other supplement.

Overnight incubation. tissue size 1x1 cm squares. I minced tissue slice with sterile scissors.

Hi Muhammad,

Did you have any success with cells adhering?

I personally have not done this for human sections, only from mouse aorta by explant culture in a media called Amniomax (Invitrogen) If interested, that is detailed in my circ res paper - there will be some obvious differences in your model and I think there are probably many references/ techniques out there for these types of dissociation/ digestion models - I've tried with limited success that way but found explant outgrowth (in mice) to be easier. Sorry I can't be of more help on that.

Best,

S

Hi Scott,

Thanks. I haven´t checked yet. Cells are sitting in the culture flask. May be one week after I will have plan for that experiment.

//Halim

Hi Scott,

It seems to me your protocol working fine. Thanks for your help.

//Halim