http://www.protocol-online.org/forums/index.php Fri, 20 Nov 2009 15:50:35 -0800 360 http://www.protocol-online.org/forums/index.php?showtopic=11701
I've got a bit of a quandary and I'd like to hear your input. My
roommate bakes bread, and I do cell culture at the lab. I know that
some labs have experienced yeast contamination of their cell lines,
attributed to baking or brewing at home.

Do you have any particular tips, other than stringent good lab
practice, that can really help prevent any contamination? I can't
prohibit her from baking (nor do I want to), but are there any special
precautions that she/we can take at home?

And from your experience: are there any of you who do bake bread at
home, without problems at work? (am I worrying overly much?)

Thanks in advance,
Megan]]> Fri, 20 Nov 2009 10:26:26 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11701 http://www.protocol-online.org/forums/index.php?showtopic=11689 Thu, 19 Nov 2009 23:13:34 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11689 http://www.protocol-online.org/forums/index.php?showtopic=11669
thanks so much!]]> Thu, 19 Nov 2009 02:59:54 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11669 http://www.protocol-online.org/forums/index.php?showtopic=11652 I have to start doing some experiment with this membrane protein I have. I need to express that in cells and do endocytosis experiments. After some literature search, I thought I'll use CHO cells. Any comments on how these cells are easy to handle in general, and if they are easily 'transfectable' and express recombinant proteins properly.
Thanks in advance for your time!]]> Wed, 18 Nov 2009 12:56:09 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11652 http://www.protocol-online.org/forums/index.php?showtopic=11638
I am planning to construct a puromycin kill curve for my hepatocellular carcinoma cell lines, however, looking at the protocol that I obtained online, I am not very sure of one of the steps, hopefully someone out there can enlighten me! Thanks very much in advance.

After adding different dilutions of antibiotics, we have to monitor the cells everyday until the antibiotics kill 100% of the cells. May I know how can we count the number of surviving adherent cells everyday without detaching them? Is counting cells the conventional way of doing a kill curve? If not, what is the normal way of doing this? Is it roughly estimate the percentage of surviving cells?

Besides, has anyone tried using puromycin on HCC cell lines e.g. SNU-398, SNU-182, HuH-7 etc. before? May I know roughly the optimal effective concentration of puromycin for these cell lines?

Million thanks!]]> Wed, 18 Nov 2009 04:11:49 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11638 http://www.protocol-online.org/forums/index.php?showtopic=11624 For an iodide uptake assay, I need to incubate adherent cells for 1 hour at 37C in HBSS with 10 mM Hepes and 10 µM NaI.
At the end of the incubation period, all the cells are round and detach leaving me with very little cells after washing if any at all. I don't know (yet) if the cells that detach are dead or not.
I've tried adding 5% BSA to the buffer but it doesn't help. My HBSS is calcium and magnesium free but I doubt this can have such a dramatic effect (and this is currently being tested!)?
Any idea of what to try next? I would rather not coat the plate with polylysine if at all possible !
Thanks in advance for your input!

Cheers!
]]> Tue, 17 Nov 2009 16:42:38 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11624 http://www.protocol-online.org/forums/index.php?showtopic=11613 Has anyone observed changes in cell morphology (cancer cells) when cultured in DMEM and DMEM/F-12? Is there any reason for this? Thanks! Tue, 17 Nov 2009 10:26:40 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11613 http://www.protocol-online.org/forums/index.php?showtopic=11606
we try to treat eukaryotic cells with some antibiotics. Now it seems like some of the antibiotics (aminoglycosides) aren't properly in the cells, so we are looking for some stuff to permeabilize the cells. Any good ideas? Links?

Thx lots for your help]]> Tue, 17 Nov 2009 07:18:17 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11606 http://www.protocol-online.org/forums/index.php?showtopic=11546
I'm trying to use HUVEC for MAPK signaling assay.

Before I stimulate them with H2O2, I serum starved them in RPMI + 1% FBS for 16 hrs, but I'm losing large amount of cells.
I collected the lysis after stimulation and 2x wash with PBS, and I got around 50ug of protein per 90mm dish...
I was expecting a lot more...I think I lost too many cells during starvation and stimulation.

I heard HUVECs are hard to serum starve, I was wondering if anyone has any advice on this problem?

Thanks in advance for your help!]]> Sat, 14 Nov 2009 19:22:13 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11546 http://www.protocol-online.org/forums/index.php?showtopic=11519 ]]> Fri, 13 Nov 2009 07:50:58 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11519 http://www.protocol-online.org/forums/index.php?showtopic=11516

I want to scale up my U-937 suspension cell line. My problem is, that the cells sink to the bottom of the flask, grow to confluency (like adherent cells, but they dont attach to the surface), and that point their division ends.

Should I use a shaker to my U-937 to produce more cells? Or simply escalate the growing surface? Any other idea?



Thanks for any reaction]]> Fri, 13 Nov 2009 06:49:02 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11516 http://www.protocol-online.org/forums/index.php?showtopic=11490
1. Improve adherence of 293 cells to an extent that is at least as good as poly-lysine (gelatin does not work very well).

2. Does not strongly compete with surface protein to be labeled with biotin.

3. Does not strongly activate signaling pathways such as integrin signal as the 293 cells will be assayed for activation of another signaling pathway that have cross-talks with integrin signaling. This sounds very hard as a lot of the coating molecules such as fibronectin and collagen would activate integrin signaling, although I'm not sure to what extent they do so. Could someone lend their expertise on this?

I also noticed that someone mentioned coating their plates with FBS in this forum. How does this work?

Any suggestions/protocols would be highly appreciated!

]]> Thu, 12 Nov 2009 08:32:17 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11490 http://www.protocol-online.org/forums/index.php?showtopic=11457 also which fixative in your experience is the best to be used for IHC????]]> Tue, 10 Nov 2009 21:11:12 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11457 http://www.protocol-online.org/forums/index.php?showtopic=11444
we are beginning to use Transwell culture plates to establish an air-liquid interface for culturing RPMI-2650, nasal epithelial cells (Bai et al. J. Pharm. Sci. 97: 1165-1178). The principle is to seed the cells in the upper chamber, let them attach for 24h, then aspirate the medium. The cells will receive all the nutrients they need from the lower chamber and they will differentiate and polarize. At the final stage the cell confluency should avoid any leakage from the lower chamber.

But we have no experience with the technique and some small tricky questions arise.

When we aspire the medium, we produce a scratch in the culture. We have tried with glass Pasteur pipette and with Gilson pipettes. The empty area is only very slowly occupied by this kind of cells. Is there any system for avoiding it? Either a better aspiration system or a different approach for emptying the chamber.

I will appreciate your help.

Thank you!


]]> Tue, 10 Nov 2009 08:55:10 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11444 http://www.protocol-online.org/forums/index.php?showtopic=11426 A) has to make a choice to migrate to one of two different other cell types (X and Y). Ideally, this would be carried out in such a way that cells XandY cannot move or migrate in response to each other, and that the cells cannot mingle with each other.

In my dream situation, I would have cell type A in the center of a chamber with X on one side and Y on the other, separated by some time of physical barrier that allows secreted factors to pass through. I know that developmental biologists have utilized similar types of assays to model migration and fate-mapping during development, but I'm not having any luck finding this right now. Does anyone have any suggestions?

thanks!]]> Mon, 09 Nov 2009 08:09:48 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11426 http://www.protocol-online.org/forums/index.php?showtopic=11414
I plan to prepare conditioned media from tumor cells for later use in culture on stroma cells to perform different assays. I was told to add protease inhibitor to the conditioned media before freezing it at -80 C. That surprises me because I would add protease inhibitor if I wanted to extract proteins but not when using it again in culture, I think proteases inhibitor could interfere with the effect the conditioned media will have or not when I will use it in culture. Anybody has an idea if I shoud effectively add protease inhibitor or not?
thanks a lot for your help]]> Sat, 07 Nov 2009 15:16:02 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11414 http://www.protocol-online.org/forums/index.php?showtopic=11406 thanks a lot]]> Fri, 06 Nov 2009 12:58:25 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11406 http://www.protocol-online.org/forums/index.php?showtopic=11378 I want to stain my cell cultures with a technique commonly used in histology. Since I can not find any reference of this I wonder: do histological techniques such as hematoxilin-eosin staining work in PFA fixed cells? On the other hand, I can't think of any reason why it should not work.
Has anybody ever done this?
Thank you!]]> Thu, 05 Nov 2009 07:05:56 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11378 http://www.protocol-online.org/forums/index.php?showtopic=11377 I am using well plates, and am unsure if I should add the drug directly to each well containing medium (e.g., a 1:100 dilution of pre-diluted drug in DMSO to DMEM), or I have also read a proper way to do it is make a stock solution of drug in the medium using a 1:1000 drug:DMEM dilution.
If anyone has experience, please help. These are kinase inhibitors.]]> Thu, 05 Nov 2009 07:01:10 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11377 http://www.protocol-online.org/forums/index.php?showtopic=11343 I have to perform cell-based proteasome Glo assay on primary cerebellar granular neruons. They grow at their best on poly-D-lysine, which in low amount can by itself stimulate proteasome activity. Does any of you know what else I can successfully grow my neurons on in order to make the assay? Many thanks for advice!]]> Wed, 04 Nov 2009 03:58:00 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11343