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Topics I've Started
Amount of RNA (ng) for good ct values.
06 February 2013 - 10:34 AM
I was wondering what is a good amount of RNA to use to get good ct values? I have been using 50ng/ul (total 250ng) per reaction and I am getting slightly high ct values. Any advice would be appreciated.
Centrifugation speeds for cells.
05 February 2013 - 10:58 AM
What speed do you guys use to spin down your cells? Is there a rule of thumb for the different types of cells in terms of centrifugation speed?
I am having a problem with my cell count and I am not sure if the spinning down process is shearing my cells. Last night I counted my cell suspension with a hemocytometer and it came out to 1.5*10^6 cells but the FACS analysis reported a much lower number (around 10^4 cells). I know I will lose cells during the staining process and during the analysis but going from 10^6 to 10^4 is huge loss. Any advice will be appreciated.
Thanks
I am having a problem with my cell count and I am not sure if the spinning down process is shearing my cells. Last night I counted my cell suspension with a hemocytometer and it came out to 1.5*10^6 cells but the FACS analysis reported a much lower number (around 10^4 cells). I know I will lose cells during the staining process and during the analysis but going from 10^6 to 10^4 is huge loss. Any advice will be appreciated.
Thanks
Cell attachement occuring after 48 hours, not 24 hours.
01 January 2013 - 02:45 PM
So I am working with epithelial cells (low passage 2-3) that do not fully attach after 24 hours. There's always about 80-90% of the cells that remain floating after 24 hours. However, after 48 hours most if not all cells attach and grow perfectly fine. A colleague says it could be mycoplasm contamination but I am only experiencing this problem with this cell line (I have no idea what mycoplasm contamination looks like). Has anyone experience something like this in the past?
I was told that the primary cells went through a rough time during their isolation. They were treated with water instead of pbs by mistake and the sorted cells took a long time to grow.
I was told that the primary cells went through a rough time during their isolation. They were treated with water instead of pbs by mistake and the sorted cells took a long time to grow.
Quick question about making culture media.
26 December 2012 - 06:16 PM
I was wondering if a slight difference is serum concentration (say 9.7% vs 10%) and supplements matters when making culture media. The media that I use the most is MEMa plus 10% ESFBS and 1% supplements. The way I make it is by removing 50mls from a 500ml bottle and add 50ml of ESFBS, 5ml of glutamine, 5ml of PenStrep, 500ul of 2-Mercaptoethanol and 5ml of NEAA. I was taught to make it this way and haven't had any problems growing cells but does the 15ml make any difference? Do you guys make your media by removing and adding the exact amount?
Procedures to replace vial of cells and passage number.
16 December 2012 - 02:21 PM
Could anyone give me brief explanation of the process in replacing a vial of cells when you plate it?
Do you grow the vial on a plate to ~80% confluency, trypsinize the cells, deactivate trypsin with medium, pellet the cells, resuspend cells in small volume (200ul) of medium, remove a small amount (~50ul) of cells for freezing and plate the remaining cells?
Also, how does passage numbering work? When exactly does it increase?
Do you grow the vial on a plate to ~80% confluency, trypsinize the cells, deactivate trypsin with medium, pellet the cells, resuspend cells in small volume (200ul) of medium, remove a small amount (~50ul) of cells for freezing and plate the remaining cells?
Also, how does passage numbering work? When exactly does it increase?
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