Hello,
I'm currently navigating the intricacies of preparing A549 cell pellets for Atomic Absorption Spectroscopy (AAS) analysis. Here's a brief overview of my process:
- Treatment: A549 cells were exposed to sodium arsenite concentrations (5µM, 10µM, 20µM) for 24 hours.
- Incubation: An extended period of 48 hours followed.
- Results: Post-washing with 800 µl PBS, each pellet revealed a robust count of 6 to 8 million cells.
However, a challenge emerged during the digestion phase. Utilizing a 1:1 HNO3/H2O2 solution, the samples exhibited unexpected effervescence in the thermomixer. While I'm considering adjusting the volume of the digestion solution, it's paramount that the integrity of cell digestion isn't compromised.
How much digestion solution would you recommend for the process? I've used 1 ml of the 1:1 HNO3/H2O2, but it seems like it is too much, since of the effervescence.
Dear Shang:
Effervescence is due to the vigorous formation of CO2 and O2. You may do the digestion in steps.
First, digest the cell pellets with conc HNO3 (eg. 0.5 ml) around 90-100 C for a couple of hours.
If you don't get a clear solution, add H2O2 in steps, e.g. 100 uL at a time.
Dilute the solutions and measure them by AAS.
You may have to play a bit with the volumes of HNO3 and H2O2.
Best wishes,
Nimal
Reduce the Volume that's means since you've noticed that 1 ml of the 1:1 HNO3/H2O2 solution seems to be too much, you can try reducing the volume. You can start with a lower volume, like 0.5 ml, and gradually increase it if necessary. This may help control the effervescence.
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