From hps4+ from pitt.edu Tue Aug 19 11:32:30 2008 From: hps4+ from pitt.edu (Hilary Stevenson) Date: Tue Aug 19 13:57:09 2008 Subject: [Cell-biology] Poly-D-Lysine and UV Light Message-ID: <001f01c90219$2cc3b0b0$43ba3182@Icarus> When I was originally trained in the use of poly-d-lysine when coating tissue culture plates and cover slips, I was told that UV light depolymerized poly-d-lysine. Is this true? I cannot find any concrete confirmation of this online, and I was wondering if it is really important to not leave them under UV light in the hood. Hilary Stevenson University of Pittsburgh Hps4@pitt.edu From Annie.Lauzier from USherbrooke.ca Wed Aug 20 12:26:31 2008 From: Annie.Lauzier from USherbrooke.ca (Annie Lauzier) Date: Thu Aug 21 11:58:20 2008 Subject: [Cell-biology] Verifying levels of p-smad Message-ID: <1219253191.48ac53c792f60@www.usherbrooke.ca> Hi, This is a general question on p-smad (2/3) detection by western. I am trying to verify the effect of different conditions on smad phosphorylation levels in fibroblasts (synoviocyte-like). I have tried by western blot with 2 different antibodies with tgfB induction as a positive control. I have bands (good), but there is no significant difference in the presence or absence of tgfB (strange - Smad phosphorylation should be increased in my cell type). I have tried different times from 15 minutes to 90 minutes after addition of tgfB. Compared to the litterature, I seem to have higher levels of p-smad in the normal condition, even after starving the cells for 24H. The bands seem to be at the expected MW. Any help would be greatly appreciated... From joannecdaniels from googlemail.com Thu Aug 21 05:57:48 2008 From: joannecdaniels from googlemail.com (Joanne Daniels) Date: Thu Aug 21 11:58:30 2008 Subject: [Cell-biology] EGF formulation for cell culture Message-ID: <9ef7a5a90808210357l7871a14dn56feb01a34bb947a@mail.gmail.com> Hello everyone, We add EGF supplement to our media when feeding cells every second day. The EGF we use is a stock made up to 10ug/ml with water, and we have been storing this stock at 40oC. I have however recently found out though that EGF is supposed to be frozen and made up fresh for each use. We have not had a problem storing the EGF at 4oC for over a month though - can anyone else tell me what they do in their group? Perhaps our EGF is degrading in the fridge, and we are effectively using a much lower concentration than we think we are! thanks for your help, Joanne From szeev from bgu.ac.il Wed Aug 27 08:28:51 2008 From: szeev from bgu.ac.il (Zeev Silverman) Date: Wed Aug 27 12:30:53 2008 Subject: [Cell-biology] extracellular mitochondria Message-ID: Using a pre-embedding approach, I'm labeling cells (2 cell line) fixed with paraformaldehyde/gluteraldehyde with an antibody against a mitochondrial protein, in the presence of 0.02% triton-X-100. The cells are then osmicated, dehydrated and embedded (spinning the cells at 1,200 rpm for 5 min. each time) in araldite. In the TEM, many of the labeled mitochondria are outside the cells. The cells themselves look good and the membranes are well preserved. Does anyone know why I consistently get this result of ejected mitochondria? Thanks, ZS Ze'ev Silverman, Ph.D. Assoc. Prof. & Chairman Department of Morphology Faculty of Health Sciences Ben-Gurion University P.O. Box 653 Beer Sheva 84105 Israel Phone: 972 8 647-7307 Fax: 972 8 647-7627 http://fohs.bgu.ac.il/brain/ From padma_channa from yahoo.com Fri Aug 29 11:53:50 2008 From: padma_channa from yahoo.com (Padma Channavajhala) Date: Sat Aug 30 11:31:17 2008 Subject: [Cell-biology] Proliferation assays in Co-culture system Message-ID: <229300.53869.qm@web50111.mail.re2.yahoo.com> Hi, I am trying to assess the proliferation of Endothelial cells which are grown in the same dish as a stromal layer. (Can't use inserts to grow endothelial system since for our purposes they need to be together this way)... Now I want to know if anybody had done a) proliferation assays of endothelial cells in this set up.. b) in our assays we grow endothelial cells for 3 days after adding them to stromal layer.. c) If I consider Brdu assay: how long i need to pulse these cells with Brdu? Followed by BrDu can I separate endothelial cells with let's say anti-CD31 antibody conjugate to Dynal beads? d) after I separate out these cells with Dynal beads, are these activated? Can I grow these separated cells? can somebody give me their opinion or suggestion or if they have used this sort of experiment please share your experience.. Thanks