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Why THP-1 cells won't attach after PMA stimulation?
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+ Thp-1
+ Macrophage
+ Culture media
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Marlon Izzo
Why THP-1 cells won't attach after PMA stimulation?
Check if the medium is calcium free. Calcium is needed for attachment. Alternatively, check if the cell culture dish is meant for tissue culture (coated). If you do fresh isolation, check you're picking up right fractions from the gradient.
Check if the medium is calcium free. Calcium is needed for attachment. Alternatively, check if the cell culture dish is meant for tissue culture (coated). If you do fresh isolation, check you're picking up right fractions from the gradient.
Maybe the PMA concentration is too high? I set the concentration of 50ng/mL, 100ng/mL, and 150ng/mL. Among them, 50ng/mL adheres the most, and 150ng/mL does not adhere at all.
Maybe the PMA concentration is too high? I set the concentration of 50ng/mL, 100ng/mL, and 150ng/mL. Among them, 50ng/mL adheres the most, and 150ng/mL does not adhere at all.
Media pH etc. L-glutamine problem is very common. Try fresh.
FCS quality
Plastic should be for adhesive cultures. (viability tested?)
infection? (mycoplasma contamination)
CO2 incubator problems (how are other cells?)
cellular stock was thawed and frozen again
So try to test viability of your cells (trypan blue or PI), correct concentration of PMA (usually stable for years). 100ng/ml correspond to 100ug/L or 10-7 g/L for rat primary peritoneal macrophages concentration above 10-8 g/L are toxic due to massive ROS production. In same time under this conditions macrophages attache to plastic very well! Optimization of PMA concentration could be done http://www.ncbi.nlm.nih.gov/pubmed/17334670
Media pH etc. L-glutamine problem is very common. Try fresh.
FCS quality
Plastic should be for adhesive cultures. (viability tested?)
infection? (mycoplasma contamination)
CO2 incubator problems (how are other cells?)
cellular stock was thawed and frozen again
So try to test viability of your cells (trypan blue or PI), correct concentration of PMA (usually stable for years). 100ng/ml correspond to 100ug/L or 10-7 g/L for rat primary peritoneal macrophages concentration above 10-8 g/L are toxic due to massive ROS production. In same time under this conditions macrophages attache to plastic very well! Optimization of PMA concentration could be done http://www.ncbi.nlm.nih.gov/pubmed/17334670
Check if the medium is calcium free. Calcium is needed for attachment. Alternatively, check if the cell culture dish is meant for tissue culture (coated). If you do fresh isolation, check you're picking up right fractions from the gradient.
Check if the medium is calcium free. Calcium is needed for attachment. Alternatively, check if the cell culture dish is meant for tissue culture (coated). If you do fresh isolation, check you're picking up right fractions from the gradient.
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Did you find the reason? We seem to have the same problem.
Did you find the reason? We seem to have the same problem.
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Maybe the PMA concentration is too high? I set the concentration of 50ng/mL, 100ng/mL, and 150ng/mL. Among them, 50ng/mL adheres the most, and 150ng/mL does not adhere at all.
Maybe the PMA concentration is too high? I set the concentration of 50ng/mL, 100ng/mL, and 150ng/mL. Among them, 50ng/mL adheres the most, and 150ng/mL does not adhere at all.
More
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Some ideas to test:
- Media pH etc. L-glutamine problem is very common. Try fresh.
- FCS quality
- Plastic should be for adhesive cultures. (viability tested?)
- infection? (mycoplasma contamination)
- CO2 incubator problems (how are other cells?)
- cellular stock was thawed and frozen again
So try to test viability of your cells (trypan blue or PI), correct concentration of PMA (usually stable for years).100ng/ml correspond to 100ug/L or 10-7 g/L
for rat primary peritoneal macrophages concentration above 10-8 g/L are toxic due to massive ROS production. In same time under this conditions macrophages attache to plastic very well!
Optimization of PMA concentration could be done http://www.ncbi.nlm.nih.gov/pubmed/17334670
Some ideas to test:
- Media pH etc. L-glutamine problem is very common. Try fresh.
- FCS quality
- Plastic should be for adhesive cultures. (viability tested?)
- infection? (mycoplasma contamination)
- CO2 incubator problems (how are other cells?)
- cellular stock was thawed and frozen again
So try to test viability of your cells (trypan blue or PI), correct concentration of PMA (usually stable for years).100ng/ml correspond to 100ug/L or 10-7 g/L
for rat primary peritoneal macrophages concentration above 10-8 g/L are toxic due to massive ROS production. In same time under this conditions macrophages attache to plastic very well!
Optimization of PMA concentration could be done http://www.ncbi.nlm.nih.gov/pubmed/17334670
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