How to identify beta pancreatic cells in dish of heterogenous cells?
Posted 26 July 2012 - 06:08 AM
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.
Posted 27 July 2012 - 05:00 AM
Posted 27 July 2012 - 08:27 AM
I will be using the antibody specific for beta cells such as insulin and Pdx1.
Thanks for the suggestion
Posted 29 July 2012 - 04:52 AM
Posted 29 July 2012 - 09:07 AM
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
Posted 04 September 2012 - 02:53 AM
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?
Posted 11 September 2012 - 07:05 AM
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.
Posted 11 September 2012 - 07:06 AM