http://www.protocol-online.org/forums/index.php Fri, 20 Nov 2009 13:57:54 -0800 360 http://www.protocol-online.org/forums/index.php?showtopic=11693 Hi.

When I treat cells with a compound I get an huge G2/M arrest, as assessed by flow cytometry. I want to check if the cells are arrested either in G2 or in M. How can I do this?

Is there any marker I can use to check this by immunofluorescence / flow cytometry?


Thanks for any answer.]]> Fri, 20 Nov 2009 04:29:36 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11693 http://www.protocol-online.org/forums/index.php?showtopic=11667 hello everyone, I encountered an awkard situation. I have two type of cells, one can tightly adhere to the fibronectin coated coverslips, and the other very weakly. we want to examine the f-actin and other related proteins by phalloidin staining or IF, which requires that the cells should stay on the coverslips whether they can adhere to the matrix or not. however, the nonadhensive cells often washed off when carrying out these staining protocols. of course, they say you can air the cells before fixation, but I am wondering if this treatment can damage the cell morphology and structure. I am very appreciated if anyone can give me some suggestions, thanks a lot. Thu, 19 Nov 2009 02:50:27 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11667 http://www.protocol-online.org/forums/index.php?showtopic=11637 Wed, 18 Nov 2009 03:39:36 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11637 http://www.protocol-online.org/forums/index.php?showtopic=11616 ]]> Tue, 17 Nov 2009 12:16:22 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11616 http://www.protocol-online.org/forums/index.php?showtopic=11601 I embedded a piece of tissue in OCT and snap froze the section. However, now I have decided to do a whole mount of the tissue and so I am not going to do any sectioning on it. My question is how do I remove it from the OCT? If I just let it come to room temperature can i just pick the tissue out of the liquid OCT? Will this damage my cells? And how can I remove the leftover glue from my tissue? Tue, 17 Nov 2009 02:18:31 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11601 http://www.protocol-online.org/forums/index.php?showtopic=11600 I have been assigned a task to extract a ethanolic fraction from various herbs. I have to freeze dry it and then dissolve the residue in water along with DMSO of PVP (to increasse the solubility of non-polar constituents). I have to use this in cell culture experiment.

My question is
How can i sterilize this herbal preperation........autoclaving wont be suitable as it degrade heat labile constituents and I am not sure about filtration of emulsions.]]> Tue, 17 Nov 2009 01:34:55 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11600 http://www.protocol-online.org/forums/index.php?showtopic=11599 Greeting, I'd be using CD34 Microbeads (Miltenyi) to isolate CD34 from bone marrow, unfortunately the CD34 microbeads didn't arrive yet and i'm expecting to recieve a bone marrow samples nowadays. That's mean I have to store samples in -80 or liguid nitrogen.
I'd like to know what's the best procedure to store bone marrow in -80 or liquid nitrogen?
should I just add 5% DMSO to bone marrow samples?
or
add media (RPMI) + bone marrow + 5% DMSO?
or
Add Miltenyi buffer to bone marrow and 5 % DMSO?
Anyone would like to share his/her protocol?
Thank you in advance

Best regards
Salem]]> Mon, 16 Nov 2009 22:53:10 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11599 http://www.protocol-online.org/forums/index.php?showtopic=11591
Can someone please tell me what ALL are the scientific reasons when Immunofluorescence staining is positive and Western blotting negative.
Ofcourse what I see in immunofluorescence staining is positive and not any unspecific staining. shRNA knockdown has confirmed this.
where i can read the scientific reasons for this case.
]]> Mon, 16 Nov 2009 18:24:45 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11591 http://www.protocol-online.org/forums/index.php?showtopic=11570 I an working with a human adult atrial cardiac fibroblasts cell-line (from ScienCell) and I have a lot of problems to obtain a response when stimulate them with typical pro-fibrotic stimuli (TFG-beta, ...).
Does anyone know another cell-line that goes well? (The cells have to be derived from a human adult cardiac fibroblasts cell-line; if possible, ventricular fibroblasts and, if not, atrial fibroblasts)

Thank you very much

Javi]]> Mon, 16 Nov 2009 04:16:58 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11570 http://www.protocol-online.org/forums/index.php?showtopic=11551 I've been having a little difficulty with 293 cells. They are not very adherent in the best of days (don't even require trypsin to dislodge them from the culture flasks), but it's been proving a disaster when I try to culture them in 96 well plates. We need them for RNAi experiments, so I really have to have equal amounts of cells in the wells. The problem is that at the washing steps, the ice-cold PBS simply washes patches off, no matter how careful I am. And I've been careful: adding the PBS dropwise onto the walls of the cells... it's taking a lot of time to do 20-30 samples, let me tell you , and I'm still loosing cells.
(Strangely there's no such problem when I tried it on bigger plates; the only problem is, of course, that the reagents are a tad expensive to conduct the experiments in 6 well format.)
I thought about using EtOH or something similar to fix the cells onto the plastic, but I'm not really sure if it would work, and if it would interefere with the lysis, RNA isolation and qPCR. (I'm very new to cell cultures.)
I'm sure people use these cells in similar settings - please give me a few pointers what I could or should try.
Any help is much appreciated.]]> Sun, 15 Nov 2009 04:44:55 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11551 http://www.protocol-online.org/forums/index.php?showtopic=11545
And NAO is the same that 10-Nonylacridine orange bromide?]]> Sat, 14 Nov 2009 11:44:31 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11545 http://www.protocol-online.org/forums/index.php?showtopic=11536 This is for all of you who do hormone treatments for mammalian cells. I know some people (seems like it is the majority) synchronize their cells before treatment, but I, personally, don't and I do not think this is a crucial point unless you are particularly interested in cell-cycle regulation or cell-cycle-related aspects of the hormone under study. I would appreciate if you guys write some comments if synchronization is otherwhise important.]]> Fri, 13 Nov 2009 16:19:18 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11536 http://www.protocol-online.org/forums/index.php?showtopic=11520
I need a mitochondrial probe that is not mitochondrial membrane potential-dependent. Can someone help me? I want to estimate if there is a bigger number of mitochondria. However I don't want to use probes that are potential-dependent.

Thanks in advance.]]> Fri, 13 Nov 2009 07:56:49 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11520 http://www.protocol-online.org/forums/index.php?showtopic=11475
I have found plastic coverslips that have the same refractive index of glass and are the same thickness, but I have heard that plastic can autofluoresce and create quite poor images. The other problem is that although these coverslips are plastic they are not polystyrene, so they still might not work for my cell type.

I have tried chamber slides (which also are a different kind of plastic) and my cells differentiate relatively well but still not great.

Another alternative is to try coating glass cover slips with something and see how that goes.

I was wondering if anyone out there has had experience with this sort of thing or can offer advice on how well plastics work for microscopy or has another suggestion.

Thanks.]]> Wed, 11 Nov 2009 13:51:09 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11475 http://www.protocol-online.org/forums/index.php?showtopic=11463 Are all floating cells in a cell culture dish dead? will the floating cells get adhered to the dish surface after sometime? Wed, 11 Nov 2009 06:37:36 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11463 http://www.protocol-online.org/forums/index.php?showtopic=11450
First of all, let me tell you what I usually do. I grow cells in coverslips inside 6-well plates. Then, I incubate the cells with medium treated with death-inducing drugs and then I do the immunofluorescence. The first step of my protocol consists in discarding the medium and wash the cells with PBS. However, death cells dettach from the plate/coverslips and, consequently, they are thrown off. When I finish the procedure I have a small representation of death cells.

I could do cytospins for the medium with death-cells, but, in this case I would only get death cells in the assay...

What can I do? This week I remembered something: gather the medium with death-cells, trypsinize the attached cells and do all the procedure in suspension and, in the end, do a cytospin. However, I loose a lot of death-cells in centrifugations...

What should I do?]]> Tue, 10 Nov 2009 10:50:59 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11450 http://www.protocol-online.org/forums/index.php?showtopic=11419 myBD protein can become blue in Z BUFFER/X-GAL, who can told me how to do next? if i want to delete the function domain, how many amino acid should be deleted? thanks. Sun, 08 Nov 2009 18:57:21 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11419 http://www.protocol-online.org/forums/index.php?showtopic=11418 all the best]]> Sun, 08 Nov 2009 10:02:50 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11418 http://www.protocol-online.org/forums/index.php?showtopic=11384
Thanks very much
]]> Thu, 05 Nov 2009 12:55:10 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11384 http://www.protocol-online.org/forums/index.php?showtopic=11327 Can anyone recommend a company which would provide a large pore size (12 uM) for cell migration assay? As the largest pore size available for transwell (Corning) is 8 uM. Thank you very much. Tue, 03 Nov 2009 17:33:54 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11327