Cell line died; unknown cause... - (Oct/03/2008 )
QUOTE (Rhombus @ Nov 26 2008, 02:35 PM)
Oh dear,
Mycoplasma testing using PCR is a waste of time...The ATCC, the world's expert and largest supplier of commercial cell lines NO LONGER USE PCR ....because it is TOO UNRELIABLE....it gives too many FALSE NEGATIVES. I try to convince all the researchers in my Institute to use the Agar Growth test in combination with Hoescht staining....both methods approved by the FDA.....PCR is not FDA approved....what does that tell you.......SO MYCOPLASMA MAY STILL BE A POSSIBILITY.
Second...... FYRITE your CO2 Incubators regularly. If the displays says 5 % DO NOT BELIEVE IT.
Thirdly ....cell lines should be routinely grown WITHOUT ANTIBIOTICS. Why are you using Antibiotics for these cells......Cryptic contaminations could be present.......If you use Antibiotics, then you should regularly leave them out for a couple of passages, to see if Cryptic contaminations reveal themselves.
Fourthly, As lost in the lab CORRECTLY mentioned, some cell lines have a definite life span. You should regularly check your cells for markers that define them. You should then go back to your master bank and revive a LOW PASSAGE vial to continue your work
Fifthly, as these cells are suspension cells, what speed do you spin your cells at. THIS IS VERY IMPORTANT AS HIGH SPEEDS CAN DAMAGE CELLS.
Hope this may be useful
Rhombus
Mycoplasma testing using PCR is a waste of time...The ATCC, the world's expert and largest supplier of commercial cell lines NO LONGER USE PCR ....because it is TOO UNRELIABLE....it gives too many FALSE NEGATIVES. I try to convince all the researchers in my Institute to use the Agar Growth test in combination with Hoescht staining....both methods approved by the FDA.....PCR is not FDA approved....what does that tell you.......SO MYCOPLASMA MAY STILL BE A POSSIBILITY.
Second...... FYRITE your CO2 Incubators regularly. If the displays says 5 % DO NOT BELIEVE IT.
Thirdly ....cell lines should be routinely grown WITHOUT ANTIBIOTICS. Why are you using Antibiotics for these cells......Cryptic contaminations could be present.......If you use Antibiotics, then you should regularly leave them out for a couple of passages, to see if Cryptic contaminations reveal themselves.
Fourthly, As lost in the lab CORRECTLY mentioned, some cell lines have a definite life span. You should regularly check your cells for markers that define them. You should then go back to your master bank and revive a LOW PASSAGE vial to continue your work
Fifthly, as these cells are suspension cells, what speed do you spin your cells at. THIS IS VERY IMPORTANT AS HIGH SPEEDS CAN DAMAGE CELLS.
Hope this may be useful
Rhombus
First, we are a huge lab that has one person checking for mycoplasm once in a while...I'm not into mycoplasm so I just assumed that they were using the best method. I will mention your comments to the persons involved.
Second, I'm not the responsible person for the CO2 supply...but I guesh it's checked regularly. I will request!
Thirdly, we are obligated to use antibiotics...so that isn't an option I'm afraid (although I agree with you)
Fourtly, I know some cells have a definite life span but I could not find one for this cell line...
Fifthly, we spin our cells at 1500-1800 rpm in a huge centrifuge (50ml tubes) (don't know the g's)
Thanks for your comments.
-Bindi-
QUOTE (Bindi @ Dec 10 2008, 08:42 AM)
QUOTE (Rhombus @ Nov 26 2008, 02:35 PM)
Oh dear,
Mycoplasma testing using PCR is a waste of time...The ATCC, the world's expert and largest supplier of commercial cell lines NO LONGER USE PCR ....because it is TOO UNRELIABLE....it gives too many FALSE NEGATIVES. I try to convince all the researchers in my Institute to use the Agar Growth test in combination with Hoescht staining....both methods approved by the FDA.....PCR is not FDA approved....what does that tell you.......SO MYCOPLASMA MAY STILL BE A POSSIBILITY.
Second...... FYRITE your CO2 Incubators regularly. If the displays says 5 % DO NOT BELIEVE IT.
Thirdly ....cell lines should be routinely grown WITHOUT ANTIBIOTICS. Why are you using Antibiotics for these cells......Cryptic contaminations could be present.......If you use Antibiotics, then you should regularly leave them out for a couple of passages, to see if Cryptic contaminations reveal themselves.
Fourthly, As lost in the lab CORRECTLY mentioned, some cell lines have a definite life span. You should regularly check your cells for markers that define them. You should then go back to your master bank and revive a LOW PASSAGE vial to continue your work
Fifthly, as these cells are suspension cells, what speed do you spin your cells at. THIS IS VERY IMPORTANT AS HIGH SPEEDS CAN DAMAGE CELLS.
Hope this may be useful
Rhombus
Mycoplasma testing using PCR is a waste of time...The ATCC, the world's expert and largest supplier of commercial cell lines NO LONGER USE PCR ....because it is TOO UNRELIABLE....it gives too many FALSE NEGATIVES. I try to convince all the researchers in my Institute to use the Agar Growth test in combination with Hoescht staining....both methods approved by the FDA.....PCR is not FDA approved....what does that tell you.......SO MYCOPLASMA MAY STILL BE A POSSIBILITY.
Second...... FYRITE your CO2 Incubators regularly. If the displays says 5 % DO NOT BELIEVE IT.
Thirdly ....cell lines should be routinely grown WITHOUT ANTIBIOTICS. Why are you using Antibiotics for these cells......Cryptic contaminations could be present.......If you use Antibiotics, then you should regularly leave them out for a couple of passages, to see if Cryptic contaminations reveal themselves.
Fourthly, As lost in the lab CORRECTLY mentioned, some cell lines have a definite life span. You should regularly check your cells for markers that define them. You should then go back to your master bank and revive a LOW PASSAGE vial to continue your work
Fifthly, as these cells are suspension cells, what speed do you spin your cells at. THIS IS VERY IMPORTANT AS HIGH SPEEDS CAN DAMAGE CELLS.
Hope this may be useful
Rhombus
First, we are a huge lab that has one person checking for mycoplasm once in a while...I'm not into mycoplasm so I just assumed that they were using the best method. I will mention your comments to the persons involved.
Second, I'm not the responsible person for the CO2 supply...but I guesh it's checked regularly. I will request!
Thirdly, we are obligated to use antibiotics...so that isn't an option I'm afraid (although I agree with you)
Fourtly, I know some cells have a definite life span but I could not find one for this cell line...
Fifthly, we spin our cells at 1500-1800 rpm in a huge centrifuge (50ml tubes) (don't know the g's)
Thanks for your comments.
Dear Bindi,
We have 220 staff in our Institute and use very many cell lines. We use an independent contractor who are experts to do our testing....no vested interests at all. They to do not recommend PCR detection
CO2 levels should be monitored weekly as infra -red CO2 sensors do drift over time, thus adding another variable into experiments. All TC researchers are taught how to fyrite their incubators as they have a vested interest in having them perfect. All cell papers that I read state in the "materials and methods" sections that " cells were grown in a humidified, CO2 Incubator set at 5%".
1500 -1800 rpm : This is far too fast for just general TC sedimentation of cells for say passaging. We 100-120g. Again using g is very important as this is the SI unit unit for centrifugation and takes into account the rMAX of the specific rotor you are using.
Just a few comments that I think are important
Kindest regards
Rhombus
-Rhombus-