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roxx_gurl's Content

There have been 17 items by roxx_gurl (Search limited from 22-February 19)


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#169357 After centrifuge in serum-free media, forming cell sheet, instead of pellet in t

Posted by roxx_gurl on 11 July 2014 - 04:54 AM in Tissue and Cell Culture

Oh 

 

Yes. It forming cell floated instead of cell pellet sink to the bottom after centrifuge.  I have experienced this before using human gingival epithelium (normal) from Cell N Tec and using their culture media, I thought they must have differentiated due to confluency and maybe because of serum-free media. 

 

Btw, I tried to neutralized trypLE (can stop detachment by dilution)  with serum-containing media (maintenance culture using serum-free media), and after centrifuge, they formed cell pellet at the bottom like normal. 

 

I guess the cell pellet formation due to serum-containing media, somehow serum attach cell together and help to form cell pellet sink to the bottom,easy to manage

 

What do you think?




#169340 After centrifuge in serum-free media, forming cell sheet, instead of pellet in t

Posted by roxx_gurl on 10 July 2014 - 09:29 PM in Tissue and Cell Culture

Hi

Has anyone encounter cell sheet across vertically in the tube, rather than normal cell pellet sink to the bottom after centrifugation ? How to get the supernatant out without disturbing the cell sheet?

 

Thanks




#169105 Cell labelling

Posted by roxx_gurl on 01 July 2014 - 10:59 PM in Cell Biology Products

Hi

 

I found interesting product of cell labeling dye (PKH 26 and PKH 67, Sigma), which yes tagging on membrane lipid as well. But this article by M.N.M Walter (2010) on wound healing assay is really cool how he tagging selectively on fibroblast and HaCat keratinocytes. But I am still not sure what is the dye tagging on and how they can distinguished both cell. Sigma product sheet not detailed in term of molecular information. I am afraid it will as well co-tagging on my mix population cells, end up no selectively labeling 




#169104 3T3 fibroblast

Posted by roxx_gurl on 01 July 2014 - 10:50 PM in Tissue and Cell Culture

Hi

 

Is there anyone here has experience on using 3T3 feeder cells (fibroblast) to grow keratinocytes? and has experience on using either mitomycin  C treated or irradiated? Which one is the best in term of time consuming and re-use the feeder cell?

 

If I am thinking of purchasing, which cell lines of feeder layer is recommended?

 

Thanks




#165267 solubility of adenine

Posted by roxx_gurl on 20 February 2014 - 03:06 AM in General Lab Techniques

I also face the same problem. I did dissolved in 0.5M HCL, it dissolved in the beginning. However when I aliquoted and froze them in -20C, it precipitates. 




#165033 Keratinocyte Culture: Cells detach

Posted by roxx_gurl on 11 February 2014 - 11:22 PM in Tissue and Cell Culture

Hi, 

 

Are you using explant technique or enzymatic ? Which protease did you use? Anyway this problem is common for keratinocytes culture. Trust me I have been culturing oral keratinocytes over a year, still could not obtain sustainable cell culture. There are many factors involved, especially when you dealing with biological sample, you might need to choose your sample carefully, I mean in term of age of patient etc




#165031 Mitomycin C Treated Feeder Cell

Posted by roxx_gurl on 11 February 2014 - 09:56 PM in Tissue and Cell Culture

Hi all, 

 

 

My supervisor requested me to have some feeder cell to support my primary oral keratinocytes growth (which has been failed despite using both explant and enzymatic technique). However, I found few protocol from website and they treated cell with mitomycin C. I have no idea which product of mitomycin C should I used. I tried Sigma but the product has been discontinued. It might be helpful if anyone has treated cell with mitomycin C and produced feeder cell. 

 

However I need to optimize the protocol as well, since I am planning to treat my primary oral fibroblast with mitomycin C. Should I just purchase the feeder cell from supplier or it is not worth it to do so

 

Thanks




#163359 L-ascorbic acid Vs Ascorbate

Posted by roxx_gurl on 12 December 2013 - 09:49 AM in Tissue and Cell Culture

Thanks




#163118 L-ascorbic acid Vs Ascorbate

Posted by roxx_gurl on 04 December 2013 - 10:42 PM in Tissue and Cell Culture

Hi scientists,

 

 

Is there anyone by any chance include ascorbate in their media? I wanted to know what is difference between L-ascorbic acid and ascorbate in cell culture media? Is there any effect in term of growth of cell or one of them degrades quickly in incubater etc. 

 

Thanks




#162619 Keratinocyte Morphology in Monolayer Culture

Posted by roxx_gurl on 19 November 2013 - 07:15 PM in Tissue and Cell Culture

Hi

 

What is the origin of your keratinocytes? Is it from skin? and is it primary or cell lines? Are you using enzymatic or explant technique? To be honest keratinocytes is pain in the ass, and you need to be patient. If you are using explant technique, the round cell might be contaminated cell. Keratinocytes is specifically has the characteristic of cobblestone when they attached and grow. That is all. If regular round shape, I am afraid that is not keratinocytes. Anyway what culture media you are using now?

 

Hope it helpsrolleyes.gif




#162473 Apo Transferrin vs Human transferrin

Posted by roxx_gurl on 15 November 2013 - 03:54 AM in Cell Biology Products

Greetings,

 

What is the difference between apo-transferrin and human transferrin? I got their definition of apo-transferrin is free from iron binding while transferrin based on Sigma product is already half-saturated with iron. Once saturated with iron, i.e forming holo-transferrin it will bring iron into cell by receptor binding of the cell. I am confused why Sigma need to have two different products which in the end both will be saturated with iron and help with cell metabolism and which product is good for cell culture like keratinocytes, Can somebody help me. Thanks




#162470 Hydrocortisone vs Water Soluble Hydrocortisone

Posted by roxx_gurl on 14 November 2013 - 09:36 PM in Tissue and Cell Culture

I am culturing normal keratinocytes isolated from gingival tissues. My Greens media recipe from my supervisor consist of water-soluble hydrocortisone. She specifically stated WATER SOLUBLE hydrocortisone. However, I bought the ethanol-soluble hydrocortisone and have been using it in my culture media over a year with inconsistent growth of cell (using explant technique), now I trouble shooting and I suspect it is because of ethanol( used to dissolve hydrocortisone) in my media causing micelles and difficult for my cell to migrate out from the explant and attach to the flask. I hope my experience helps you. cool.png




#162427 Insulin bovine pancreas origin vs insulin recombinant human

Posted by roxx_gurl on 13 November 2013 - 05:43 PM in Tissue and Cell Culture

Hi all, 

 

Is anyone has experience on using these products from Sigma? My current product is insulin from recombinant human expressed in fungal produced by recombinant DNA technology in baker's yeast Saccharomyces cerevisiae and I am having problem with my cell doesn't grow. 

 

Thanks




#162426 what is the difference between water-soluble and ethanol soluble hydrocortisone

Posted by roxx_gurl on 13 November 2013 - 05:39 PM in Tissue and Cell Culture

Thanks. No wonder my keratinocytes does not grow wacko.png




#162269 what is the difference between water-soluble and ethanol soluble hydrocortisone

Posted by roxx_gurl on 10 November 2013 - 09:19 PM in Tissue and Cell Culture

Hello everybody, 

 

I have been using hydrocortisone that been dissolved by ethanol (cheaper) despite the recipe from my supervisor is water soluble ethanol. However the result of isolated cell is not consistent, I thought my media works in beginning, but more failure than success. I am troubleshooting and wanted to check if ethanol-dissolved hydrocortisone giving me the problem. Is there anyone knows what is the difference between water-soluble and ethanol soluble hydrocortisone in term of primary cell growth?

 

Thanks 




#162174 Trypsin-EDTA

Posted by roxx_gurl on 07 November 2013 - 02:41 AM in General Lab Techniques

Great website. Thanksbiggrin.png




#162164 Trypsin-EDTA

Posted by roxx_gurl on 06 November 2013 - 08:44 PM in General Lab Techniques

How to dilute from 0.25% Trypsin-EDTA (1x) to 0.025% Trypsin-EDTA (1x). I am confused what is the percentage stand for since all the method in Google only mentioned on diluting 10x to 1x trypsin-EDTA

 

Thanks





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