12 replies to this topic

[wdyeo]

Dear all,
I am wondering if there is any identification method to identify beta pancreatic cells, based on morphology for example, from a dish of heterogenous cells?
I am currently, selecting the zone of interest from a dish and do immunofluorescence on it....but it seems to be finding needle in a haystack.
Thank you.

wendy

[Curtis]

How do you do IF? on what protein/marker?

[zodiac1505]

I guess you could try staining the cells with dithizone. It is a dye which binds to zinc ions which are abundant in the islet cells and stains them red (within a couple of minutes). The non islet cells won't be stained. You could then pick the red cells out under a microscope for instance. The staining is reversible and so it shouldn't affect your analyses.

[wdyeo]

Dear Curtis,
I will be using the antibody specific for beta cells such as insulin and Pdx1.

Dear zodiac1505,
Thanks for the suggestion

[Curtis]

I am not really optimistic if there is a method to differentiate those cells from others by just their morphology. Sometimes similar cells in a cell line grow in different shapes too. Also, what I meant is that how do you grow the cells? Do you grow on a coverslip? Do you have controls? Do you mount your cells well? What mounting buffer do you use? Did you know Invitrogen has a product that enhances the intensity of your labels?If the IF labelling goes well then you must be seeing some labelled cells easily.  If you don't see any labelled cells then perhaps there is no beta cell in there? You run them on a flowcyto machine or you view under microscope? I personally prefer microscope. Don't ask me why!

[wdyeo]

Dear Curtis,
Yeah, i do agree with u...if based on morphology, there's no 100% but i would like to know at least if there's a 'hint' if there are beta cells in the culture, based on their morphology. I grow them on 100mm cell culture tissue dish. Yeah, i am using INS1 cell line, an insulinoma cell line as a control when i am doing the IF staining. I use Airvol to mount the cells.
Nope, i have to idea about that product. May i know the name of the product please? Thank you.
Yeah, me too, prefer to use microscope and flowcytometer is way to compliacted for me...hehhe

[Curtis]

ProLong® Gold and SlowFade® Gold

[wdyeo]

Hi Curtis,
Thank u very much !!!

[wdyeo]

Dear zodiac1505,
I have bought the dithiozone (DTZ) powder and make it but when i tested on INS1 cell line (rat insulinoma) which is expressing insulin, i cant detect any red cells ,..
I have incubated for 15 min and my concentration is 1g/L of DTZ, with 10ul of DTZ for every ml of media and i even increase to 1 ml of DTZ for every 1 ml of media.
Below is my protocol for making the DTZ solution.

Stock dithizone solution
1. Add 20mg of dithizone to 0.6ml of 96% ethanol in a 15ml conical tube.
2. Mix thoroughly by vortexing. Stain will not dissolve completely until after step 3
3. Add 1-2 drops of ammonium hydroxide and mix.
Dithizone will go into solution and turn bright orange

Final dithizone solution
1. Add 0.3ml of the stock dithizone solution to a volumetric flask.
2. Add phosphate buffered saline to a final volume 100 ml.
3. Mix and slowly add 1N HCl to adjust pH to 7.4. and filter 0.2 µm. Dispense this solution into 5ml aliquots and store at –20°C.

May i know what shall i do?

Thank you.

wdyeo

[wdyeo]

Dear All,
I have found out today that the protocol to make DTZ is working...just because my cell line do not contains zinc
Thank u ~

[Curtis]

Great news

[zodiac1505]

Hi wdyeo

Sorry for the late reply. Hadn't come online for a few days. Do you mean the staining works but that Ins-1 cells do not express Zn and that is why you didn't see any red cells? I had done the staining on pancreatic tissues but have never tried it on any of the insulinoma cell lines. I used to work with both INS-1E and NIT-1.

[zodiac1505]

And as far as I remember, I just dissolved the DTZ in distilled water and washed it off with distilled water. Also the staining never took longer than a couple of minutes.