http://www.protocol-online.org/forums Fri, 10 Feb 2012 12:36:24 +0000 360 http://www.protocol-online.org/forums/topic/24373-problem-with-preparing-unicellular-suspension-from-mcf7-cells/ I have a big problem with preparing a unicellular suspension from MCF7 breast cancer cell line. I need to do this because I need to establish a SCID model of human breast cancer injecting the mice with exact number of cells.
The problem is that after I take off the cells from the flask (I have tried different things: Trypsin, Acutase and also a scrap tool), centrifuge and resuspend them I can't count them because cells form floating islands and groups, which doesn't allow correct counting. The same is happening when I am using C-26 colon cancer cell line.
If anyone knows how to solve this problem please do help me-this stops my work from the very begining!]]> Fri, 10 Feb 2012 12:36:24 +0000 http://www.protocol-online.org/forums/topic/24373-problem-with-preparing-unicellular-suspension-from-mcf7-cells/ http://www.protocol-online.org/forums/topic/24366-seeking-advice-regarding-contamination/ ,

At first I am very gratef ul for your comments and advices.
I have no one except you to seek for your priceless help.
I need your advice in two issues:

1) I am in a middle of experiment using keratinocyte from ATCC, I cultured it firstly with Antibiotic then without antibiotic.
it was fine and I was doing my experiment on 24 well plate, it takes around 4 days.
the first, second and thirds day was ok, on the third day I change the medium and add new medium, and on the fourth day I saw something like sever white turbidity in 17 wells out of 24, and I cant see my cells, they are gone, not attached to the surface of my plate, nothing only in 24 hrs.
I have added the new medium using the same pippte and the same tip for all wells, and the reagents for my experiment too.
The most strange things is in another cultured 98 well plate (3 out of 98) showed the same sever turbidity, this was cultured in another day.
the rest of my plate was fine too, no turbidity and cells looks fine under microscope.
So I have took some photo using 100 X (oil immersion) as these intruders are very tiny and cant be clearly seen under 40X.


2) I am using new FBS, on culturing another type of cells (No Antibiotics too), and what I noticed that there are a lot of things floating in my medium under microscope, but not turbidity which could be seen by naked eye, I dont know if they are cell debris or debris from my new FBS itself.
so my question, to identify contamination what shall I expect ( I am not using antibiotics),
Sever turbidity??? or slight one.
Contamination occupy all my plate or minor one??
death of my cells and the first issue ( but my cells are 97% viable using trypan blue.)
I have added a photo from American National Cancer institute which describes what you can expect in contamination, A is non contaminated cultured cells, so shall I expect something like that and my cells be normal.



Thank you guys in Advance,
Best Regards]]> Fri, 10 Feb 2012 06:29:22 +0000 http://www.protocol-online.org/forums/topic/24366-seeking-advice-regarding-contamination/ http://www.protocol-online.org/forums/topic/24363-xenograft-cells-harvested-from-spleen-of-scidnod-mice/
I'm use to working with cell lines and this is the first time I am working with xenograft cells harvested from the spleen of SCID/NOD mice. I am currently practicing an Alamar blue cytotoxic assay. I bring the cells up from liquid nitrogen and plate them the afternoon before I intend to incubate them with cytotoxic drugs for 48hrs and then adding the Alamar blue and measure the florescence at 0hr and 6hrs. This is a well established protocol by the lab I am currently in. My problem is that the cells in all of my wells (including controls) die before the day when I'm suppose to add the Alamar blue. I think that maybe its my freeze thaw technique that maybe causing the cells to die very quickly. The only criticisms I am getting is that I am really gentle when I bring the cells up from liquid nitrogen. Can someone please give me some tips on how I could be bringing the cells up successfully from liquid nitrogen or does anyone have any insight on what might be the problem??

Thanks.]]> Fri, 10 Feb 2012 01:04:54 +0000 http://www.protocol-online.org/forums/topic/24363-xenograft-cells-harvested-from-spleen-of-scidnod-mice/ http://www.protocol-online.org/forums/topic/24333-3t3-l1-differenciation-and-maintenance/ Different protocols are available:
I have some questions concerning different steps:
can I use FBS for proliferation instead of calf serum?
Is it necessary to use insulin in differenciated adipocyte maintenance medium (used 2 days after the insulin media and 4 days after differentiation initiation)?
Thank you very much.

Best regards,
Fred]]> Wed, 08 Feb 2012 11:12:06 +0000 http://www.protocol-online.org/forums/topic/24333-3t3-l1-differenciation-and-maintenance/ http://www.protocol-online.org/forums/topic/24325-culturing-cells-on-a-slide/ Dear Guys ,
In the beginning, I am very grateful for any one who will read my topic.

Secondly, I want to buy some culture wares for visualization of my cells under fluorescence or phase contrast microscope.
what I found due to reading some books and online:

*There are special type of culture slide such as
http://www.bdbioscie...y=4&mterms=true

*It is untreated.
I should treat the surface with Poly L lysine or collagenase as I am working with adherent cells
but I cant know the difference between these reagent and which on is the best.

* After culturing, Does the step of fixation is necessary, or adhesion to surface of my slide is enough.
* Shall I use the ordinary glass cover on my slide or use these precoated glass covers or precoat them by myself.
http://www.bdbioscie...rue&from=dTable

finally, does these steps differe if I will use a laser scanning confocal microscope?? Does any one has experience with it and ready to share??

At the end I am deeply indebted for those who will share their thoughts, experience with me.

Best Regards ]]> Wed, 08 Feb 2012 04:13:58 +0000 http://www.protocol-online.org/forums/topic/24325-culturing-cells-on-a-slide/ http://www.protocol-online.org/forums/topic/24321-help/


I have a problem with expression of GABA receptor. Nowadays I am working with WSS-1 cell line form ATCC. Unfortunately I have problem with obtaining high expression of β-receptors in this cell line. The cell were cultured accordingly with the recommendations of ATCC. Vibality of cell after a passage were about 98%. In addition, lead the culture of cell with Geneticin (400µg/ml). But also we don’t observed high expression of β-receptors in this cell line (the Geneticin was added to 2 passage of cells). Cells cultured in medium with Geneticin showed smaller increase compared to cells in medium without geneticin. After 2 days of culture reached confluency less. Has anyone worked with this cell line? This is very important for me.]]> Tue, 07 Feb 2012 17:57:41 +0000 http://www.protocol-online.org/forums/topic/24321-help/ http://www.protocol-online.org/forums/topic/24318-can-you-freeze-thaw-dmem-and-it-still-work/
Odd question - but hear me out!
I'm currently testing the effect of some peptides on the migration of 3T3 fibroblast cells over a scratch wound.
I need to incubate my cells in DMEM media containing the peptide (which is purchased as a powder) for a couple of hours before assaying my cells.

The concentrations of the peptide in the media is really quite small (i.e. 100uM) and so I'm measuring out miniscule amounts of the peptide powder to dissolve into the media - which is leading me to believe that I'm losing some accuracy along the way.

An obvious solution would be to dissolve more powder into a larger aliquot of DMEM media = less room for error.
But I won't be using the peptide/media combination that often and I don't want to just throw it away when my media naturally degrades as the peptide is very expensive.

So I would naturally want to freeze my DMEM/Peptagon combination (in aliquots to thaw out when and as needed).

BUT - Can you freeze-thaw DMEM and it still work optimally?
I can't find anything to say you can't - so I'm guessing it's OK....]]> Tue, 07 Feb 2012 16:28:41 +0000 http://www.protocol-online.org/forums/topic/24318-can-you-freeze-thaw-dmem-and-it-still-work/ http://www.protocol-online.org/forums/topic/24291-how-to-dilute-cell-lysate-for-luciferase-assay/ Thanks
A.P.C.]]> Sat, 04 Feb 2012 17:27:37 +0000 http://www.protocol-online.org/forums/topic/24291-how-to-dilute-cell-lysate-for-luciferase-assay/ http://www.protocol-online.org/forums/topic/24281-whats-the-function-of-cholera-toxin-in-cell-culture-medium-eg-mcf10a-cells/ As the topic, does anyone know how does cholera toxin help mammary epithelial cells grow? Thanks. Fri, 03 Feb 2012 19:28:29 +0000 http://www.protocol-online.org/forums/topic/24281-whats-the-function-of-cholera-toxin-in-cell-culture-medium-eg-mcf10a-cells/ http://www.protocol-online.org/forums/topic/24267-could-the-use-of-different-fbs-affact-my-cells/ At first I am very grateful for all your help in advance.
I was using FBS from a company called CCB (cell culture biosience) http://www.nichirei.co.jp/bio/products/safc/Cell_Culture_Bioscience.html
.
then we have bought FBS from other company called biowest ( french origin).
http://www.biowest.net/eu/index.php?pg=Animal_Serum_Products_List&num_cat=1&cat=Animal%20Serum&menu=off

my question could the change of FBS affect my cells???
Does anyone has a similar experience with different suppliers for different FBS.
is it better to filter (0.2 microfiltration my biowest FBS before I aliquot it into smaller volume???
Thanks guys for your thoughts, experience and comments
Best Regards
Shimaa]]> Fri, 03 Feb 2012 01:44:43 +0000 http://www.protocol-online.org/forums/topic/24267-could-the-use-of-different-fbs-affact-my-cells/ http://www.protocol-online.org/forums/topic/24263-does-dna-have-to-be-supercoiled-for-transient-protein-expression/
Thanks!]]> Thu, 02 Feb 2012 20:39:16 +0000 http://www.protocol-online.org/forums/topic/24263-does-dna-have-to-be-supercoiled-for-transient-protein-expression/ http://www.protocol-online.org/forums/topic/24258-adscs/ I'm the new member and just started my study.
Just wanted to know if any of you have ever worked with ADSCs and do you know about their freezing medium?
helps are highly appreciated
Regards
Bita]]> Thu, 02 Feb 2012 12:49:03 +0000 http://www.protocol-online.org/forums/topic/24258-adscs/ http://www.protocol-online.org/forums/topic/24242-differences-between-j7741-and-raw-2647-murine-macrophages/
Any help or comment is much appreciated.
Thanks]]> Wed, 01 Feb 2012 11:42:19 +0000 http://www.protocol-online.org/forums/topic/24242-differences-between-j7741-and-raw-2647-murine-macrophages/ http://www.protocol-online.org/forums/topic/24224-high-glucose-for-primary-astrocyte-or-neurons/
Does high glucose concentration means 25mM of glucose or something higher? (for common cell lines, the normal glucose level should be 5mM as I remember...)

Also, for these cultures, is "high glucose" means "normal level" for them? (Since I would like to treat them with high glucose treatment, so need to choose the concentrations now...)]]> Tue, 31 Jan 2012 10:59:34 +0000 http://www.protocol-online.org/forums/topic/24224-high-glucose-for-primary-astrocyte-or-neurons/ http://www.protocol-online.org/forums/topic/24208-number-of-snap-per-hour-in-time-laps-system/
I'm going to do some time laps experiments using HONE cell line and I'm wondering how many snaps I can take per hour. More precisely I'm wondering how many fluorescence exposition i can do per hour (24h total) to avoid or have small phototoxicity. If you have any average it could be a great help.

Thanks in advance.

Thibaut]]> Mon, 30 Jan 2012 13:26:22 +0000 http://www.protocol-online.org/forums/topic/24208-number-of-snap-per-hour-in-time-laps-system/ http://www.protocol-online.org/forums/topic/24207-lymphoblastoid-cell-culture/ Hello,
2 weeks ago I started a lymphoblastoid cell culture. I am facing a problem to get them grow. I started from 5,000,000 cells in 2.5 ml RPMI (FBS, L-Glu supplemented) (protocol from another lab.). I guess most of the cells died. Now, I have only 400,000 cells. I am not sure why they are not growing properly. I know the cell density plays a crucial role. Any idea, on what can be going wrong?

If you need more info please let me know!
Thanks
Hussein
]]> Mon, 30 Jan 2012 11:10:45 +0000 http://www.protocol-online.org/forums/topic/24207-lymphoblastoid-cell-culture/ http://www.protocol-online.org/forums/topic/24204-counting-cells-dilution/
The other day I had to count cells after resuspending my cells in 4mL. I took 10uL of cells mixed with 10uL of trypan blue and loaded onto a hemocytometer...I had soo many cells I couldn't count so I diluted my cells 15 fold (3.3uL of cells + 46.7uL of media +50uL of trypan blue)..I counted around 381 cells in the grids all up so just wondering when its time to count the total amount of cells in that whole 4mL do I use the following formula.

(381*15)*2*10^4=1.143*10^8 multiplied by 4 =4.6*10^8

seeing as i diluted 15 fold i assume i multiply the total number of cells by 15 and then because theres a 1:1 ratio of trypan blue I multiple by 2 and then 10^4 becuase one set of 16 corner squares is equivalent to the number of cells x 104 / ml and then i multipled by 4 because thats how much i resuspended in...have i got this right? I think I may have got the typan blue dilution factor wrong because when I previously counted cells I never had to dilute my cells as they were countable...so i just used 2 as the dilution factor..have i got this new dilution factor right?..hope im making sense!

thanks
biology_06er]]> Mon, 30 Jan 2012 03:58:45 +0000 http://www.protocol-online.org/forums/topic/24204-counting-cells-dilution/ http://www.protocol-online.org/forums/topic/24202-problem-with-soft-agar-assay/
However, now I came across a bizzare problem. I can see many cells adhered to the plate within a few days of cell plating and growing in monolayer instead of forming typical three-dimensional colonies. I know I had no problem in pouring the agar. These are breast cancer cells. Bottom layer is 2.5 ml of 0.5% Agar Noble (in DMEM) per well and top agar is 1 ml of 0.33% Agar Noble per well (with 25,000 to 100,000 cells in MEGM).

Somebody in this forum had posted similar problem in August 2010, which got no attention or reply from anybody. Please help me out here if you guys have any thoughts.

Manish]]> Sun, 29 Jan 2012 22:27:43 +0000 http://www.protocol-online.org/forums/topic/24202-problem-with-soft-agar-assay/ http://www.protocol-online.org/forums/topic/24181-sf9-cells-lose-exponential-growth-lack-stationary-phase/
I've been maintaining shaker flasks of SF9 insect cells for the past 2.5 years using a single cell line, but recently I've been having some problems.

When I subculture the cells to 2.5x10^5 cells/mL, they typically double in 24 hours. After those 24 hours have elapsed, however, they rarely double again, and seem to enter a phase of linear growth (i.e. day 2: 5x10^5 cells/mL, day 3: 7x10^5, day 4: 9x10^5). What is also disturbing is that they have a relatively low high density maximum around 1x10^6 (when my lab could typically get around 2x10^6 cells/mL easily), and also that they do not remain at their maximum for any period of time -- while it used to be the case that the cells could be left for up to a week in stationary phase, these cells are now reaching about 9x10^5 cells/mL on average before slowly decreasing.

Let me provide some more information:

-I have been using the same cells all along, and have retrieved cells from frozen stock to make sure the passage number is not too high.
-I use grace's insect medium +10% FBS +pluronic surfactant in a shaking incubator at 100 RPM at 27.0 celsius. These parameters have not changed. They are in shaker flasks with a relatively low volume, with sealed caps. I have tested vented caps and different medium (tc-100) but nothing has changed.
-There is no visible sign of contamination, at least nothing obvious.
-I work with baculovirus, and the stocks are negative for contamination with that.
-When viewing the cells using typan blue exclusion, they seem reasonably normal/happy, and I hardly ever see a dead cell

Anyone have any ideas what could be going wrong? The help would be greatly appreciated!]]> Fri, 27 Jan 2012 21:28:24 +0000 http://www.protocol-online.org/forums/topic/24181-sf9-cells-lose-exponential-growth-lack-stationary-phase/ http://www.protocol-online.org/forums/topic/24170-dont-know-why-my-cells-are-unhealthy/
The vendor we sourced these cells from recommend growing them in Ham's F-12 + 10% FBS with double selection: 750 ug/ml zeocin and 10 ug/ml blasticidin. I do not add P/S. Our frozen stock has been in use for 3 years so I am sure they are OK. The cells come out of thaw looking healthy, but after passing them, they look terrible! Only a few adhere to the flask and if they divide at all, it is very very slow growing. To get them to grow at a normal rate, we have lowered the zeocin concentration to 400 ug/ml, but the cells still look unhealthy (several senescent/multinucleated cells) and assays produce only mediocre results.

If I remove selection, the cells grow normally. It seems that there may be some sort of negative interaction with the base medium and selection that make the cells unhappy. My colleague recently brought up CHO-M1, which she has growing in Ham's F-12 + 10% FBS (different brand & lot from the CHO-hERG above) and 100 ug/ml geneticin. They also have the same abnormal cell morphology the CHO-hERG cells have. They only thing in common between these two cell lines is the Ham's F-12 they are grown in. They are in different incubators. Between my colleague and I, we have about 10 different cell lines in culture and only the two in Ham's F-12 have this issue. We have also tried a different brand of Ham's F-12, but we did not see any improvement. For all I know, the two vendors could be sourcing their raw materials from the same place.

Any thoughts on what my problem could be? Anyone ever encounter an issue like this before? I've called the medium vendor and cell line vendor and neither have been able to help me. ]]> Thu, 26 Jan 2012 23:24:49 +0000 http://www.protocol-online.org/forums/topic/24170-dont-know-why-my-cells-are-unhealthy/