I collected PBMC and cultured the cells in full medium with 10 ng/ml PMA in order to polarize monocytes to macrophages without isolating the monocytes from PBMC. After 24h I washed and discard non-attached cells. However, the background is full of small and long particles. What are these particles and do they affect polarization? If it affects polarization how can we discard them?
Maybe bacterial contamination, you can make sure by a smear and dyeing by Gram stain.
Hello Ahmed Al-Omar,
this is all bacterial contamination in the background. Your need to discard this culture flask since bacterial growth will certainly have a negative impact and repercussions on cell growth and polarization. For subsequent isolation, you may add antibiotics to your culture media, including PenStep (commercially available Penicillin and Streptomycin cocktail). This will prevent any bacterial growth. God luck!
Jatin Chadha Thank you Dr. Jatin, the medium already contains PenStep, are you sure they are bacteria? because some of them have triangle shapes and today I take a new picture after 5 days of PMA there morphology became a circle. Could it be platelets?
Behzad Nezhadasad Aghbash There is small tiny particles in the field, and I wanted to identify what these particles are? Now everything is fine thank you for your participation.
Just looking at your micrographs, I suspect it is bacterial contamination and likely pen/strep resistant. Try gentamicin instead. But if the cultures are infected, it can be extremely difficult to clear it. It would be better to start fresh.
How do you isolate your PBMC? By Ficoll gradients?
- Badges
- Science topic
- Similar topics
- Biological Science
- Physiology
- Physiological Processes
- Stress