http://www.scientistsolutions.com/c580-cell+culture+and+tissue+culture.html Discussion on 2D and 3D cell and tissue culture, primary cells, cell lines and transformed cells, isolation and culture of particular cell types such as stem cells, lymphocytes, epithelial and endothelial cells, transfection, electroporation, cell imaging, detachment, apoptosis… Life Science Discussion en-us sci7feed@sci7.nojunkorherepleasespam.com hourly 1 2005-05-05T12:00+00:00 http://www.scientistsolutions.com/t27179-imr_32+cells.html I'm trying to grow IMR-32 cells. I have never used these cells before and am having difficulties. The cells don't seem to attach very well to the flask and grow in clumps, and as such they don't seem to be proliferating. Does anyone have any experience working with these cells? If so could you please give me some tips?
Thanks!
]]> Mon, 03 Jun 2013 02:50:14 GMT http://www.scientistsolutions.com/t26967-j774+macrophage+granulation.html I am facing some problems with regard to my J774 cells. Everytime I seed them they look fine but in 2 days they start looking highly granulated. Besides this they don't seem to adhere well to the TC flasks and come out easily . What could be the possible problem?]]> Thu, 23 May 2013 01:24:03 GMT http://www.scientistsolutions.com/t26961-sf9+cell+culture.html I am currently trying to grow Sf9 cells for baculovirus expression experiments. I am however having great difficulties. I purchased a vial of cells from Invitrogen and set them up in suspension culture, however they haven't grown at all and have suffered a massive loss of viability. So I now only have 4X105 cells/ml in 5mls media with lots of dead cells and debris around. I really don't want to have to purchase more cells and have no frozen stocks so I could do with rescuing these cells. ...]]> Tue, 21 May 2013 07:32:56 GMT http://www.scientistsolutions.com/t26821-has+anyone+been+alvetex+3d+culture+scaffold.html I am a new entrant to the world of 3d cultures. Has anyone been using the alvetex 3d culture scaffolds. If yes, how was your experience with it. Is a there a way you can image the live cells using phase contrast microscope for routine checks? and how do we the scaffolds for live cell confocal imaging?]]> Mon, 06 May 2013 06:04:37 GMT http://www.scientistsolutions.com/t26732-western+blot.html 1) May I know what type of antibody needs to be used for primary and secondary? My guess is, if my primary antibody is from a mouse, so my secondary antibody should be from mouse source as well.
2) Is it advisable to to re-use the same mebrane for other antibody for up to three times?
3)If I am to set up a knockdow...]]> Tue, 30 Apr 2013 12:38:36 GMT http://www.scientistsolutions.com/t26729-cytotoxicity+and+96+well+plan.html I want to determine the IC50 value of calcitriol (vitamin D) on my cell line but i am a bit indecisive about what my 96 well plate plan should be like.
I dissolve calcitriol in DMSO so it should be like
1-medium+DMSO
2-DMSO
3-medium+calcitriol+cell
Or it should be like;
1- medium+calcitriol+cell
2- medium+calcitriol
3- medium+cell
4- medium
Thanks,]]> Tue, 30 Apr 2013 01:45:46 GMT http://www.scientistsolutions.com/t26649-dmso+toxicity.html I have a query, i am working on cell line. I need to dissolve my toxic compound in DMSO and want to check the toxicity.
my query is how to dissolve my compound in DMSO (99.9 % pure), since both are aqueous solution how to check the solubility ?
2. DMSO is 99.9 % pure and also toxic when exposed to cells, so how to bring down the DMSO concentration from 99.9 % to 0.1 % ? How to dilute whether in medium or in water..? Then when to add my compound of interest..? ...]]> Tue, 23 Apr 2013 22:07:28 GMT http://www.scientistsolutions.com/t26579-adult+neuron+culture.html Mon, 08 Apr 2013 08:49:11 GMT http://www.scientistsolutions.com/t26306-t47d+_amp%3b+mcf7+cells+die+in+soft+agar+assay+after+3+_+5+days.html since this is my first post, please excuse any mistakes.
I am trying to set up a Soft-Agar-Assay for MCF-7 and T47D-cells. I use Agarose DNA Pure Grade in a .5% Bottomlayer and 0.35% Toplayer (containing cells) on 12-well plates. I managed to get the cells (usually 25000 or 50000 per well) to grow and form colonies, but after a couple of days, the just die. I change medium every three days. I've tried several things in order to optimize the protocoll I am using, but no...]]> Tue, 26 Mar 2013 02:19:34 GMT http://www.scientistsolutions.com/t26293-vacuolized+cells%2c+particles+in+culture.html I have the same problem accross cell lines accross incubators. I have cells fresh from ATCC, from liquid nitrogen stock, and cultures that were growing fine until now. Cells look vacuolized, stop growing, release particles into the medium. we have inspected the hood (all normal), sterilized the incubators, changed all the medium and reagents. Nothing helped. It seams to be spreading (not sure how) to all different cells lines - we grow about 20...]]> Fri, 22 Mar 2013 11:44:30 GMT http://www.scientistsolutions.com/t26289-transfection+of+a+230+kda+protein+in+hek293.html I'm trying to overexpress a protein of 230 KDa in HEK293. I have been trying for 3 months but I don't see anything on my western blot...
I use Lipofectamine, or Xtrem Gene ( supposed to be better)
I lyse with SDS2%
I use a 4-12% NuPage gel, and i transfer during 3 hours approximately
I use antibodies tthat have been validated on western before...
Any idea to optimize the protocol?
thank you very much,
Rachel
]]> Fri, 22 Mar 2013 08:00:35 GMT http://www.scientistsolutions.com/t26142-wst_1_+i+can_t+get+the+idea.html Consider that i find IC50 value of calcitriol 0,000001 mM with 36 hours incubation. Is it possible to use higher concentraion and lower the incubation time? Which one is impornant? Longer incubation time or higher agent concentraion?. In my opinon longer incubation time should be important because cells then can metabolise the agent.
Thanks a lot!!]]> Sun, 03 Mar 2013 03:35:06 GMT http://www.scientistsolutions.com/t26138-ovarian+cancer+cell+detach+and+black+dots+appear.html I seeded about 1000 cells in 24-well plate and grew them in phenol-red-free M199 medium, supplemented with 2.5% charcoal stripped FBS. It attached initially, and did grow in number. But on the third day when I checked, the cells detached and were floating, with black dots visible.
Are the cells contaminated? I am suspecting several things,
Could it be that the cell density is too low and under r...]]> Fri, 01 Mar 2013 01:42:37 GMT http://www.scientistsolutions.com/t25996-cell+culture.html I am working on SK Hep 1 cells. This is the second time in a month that the cells have got infection. They are infected with short rods. The media in the cells is turbid and has some swarming growth. I have tossed a lot of cells already. there is only one flask which has pretty low load of rods and I want to know how to treat this last flask.
I don't know what and where I'm going wrong.]]> Tue, 26 Feb 2013 17:57:26 GMT http://www.scientistsolutions.com/t25922-xtt+and+trypan+blue__ic50__.html Can someone please help me.. i'm really confused now. I've got the IC50 value of my drug from XTT assay and then i treated my cancer cell line with the IC50 value and did trypan blue exclusion method. I found that less than 10 cells were alive. From what I understand IC50 value is the concentration of the drug/sample that could inhibit 50% cell growth but then why did the IC50 value killed almost all of the cells??
Please help me..
Thanks!!]]> Fri, 22 Feb 2013 21:31:47 GMT http://www.scientistsolutions.com/t25807-fcs+concentration+to+simulate+the+serum+conc_+in+wound.html With my speculations i just don't get any further. I wonder, which concentration of FCS do you reckon as an appropriate to simulate the concentration of human serum (incl. the whole bunch of growth factors, cytokines etc.) in the wound during the wound healing process.
In the majority of studies 10% FCS is being picked, but i haven't found even one article explaining why.
You're my hope )
Many thanks in advance!!

...]]> Fri, 08 Feb 2013 07:12:48 GMT http://www.scientistsolutions.com/t25804-dapi+and+u937+cells.html i have cultured U937 cells from ATCC. i wanted to get some images of them, so i mounted slides with vectashield containing DAPI and put them under the fluorescent microscope.
when i did, the DAPI-stained nuclei looked almost as large as the rest of the cell (when viewed under visible light). is this common? i'm worried because we also culture jurkat (t-cells), and don't want to think i'm working wit...]]> Thu, 07 Feb 2013 13:06:18 GMT http://www.scientistsolutions.com/t25758-adhesion+of+primary+mouse+epithelial+cells_.html I was wondering if anyone had some advice for getting primary mouse skin epithelial cells to adhere.
I've been able to isolate the cells well enough, but when I place them in a 24 well plate with Poly L-lysine treated coverslips (which has worked great for me in the past with 293T cells), they won't adhere. Interestingly, they are also no adhereing to the bottom of the wells either so it may not just be an issue of the poly lysine.
Thanks
]]> Thu, 31 Jan 2013 12:43:21 GMT http://www.scientistsolutions.com/t25757-proper+cytotoxic+drug+handling_.html No, what I ask for is personal experience and useful tips.
I am working / will work with the following drugs;
Permetrexed, Carboplatin, Doxorubicin, Gemcitabine, Vinorelbine.
I test drug viability by regurarly kill off cell lines with known sensitivity to above drugs.
Now, the problem ...]]> Thu, 31 Jan 2013 02:30:11 GMT http://www.scientistsolutions.com/t25656-cell+counting+and+mtt+assay.html I was doing MTT assay IN HUMAN LIVER cells. but it is not working well, the results are not appropriate in control and in treated cells.
Can anyone say me how to troubleshoot..or give the procedure and calculation for cell counting for hemocytometer..
waiting fr reply
thanks]]> Sat, 26 Jan 2013 23:03:27 GMT