I've been banging my head against the wall trying to figure this out for over a few months & would greatly appreciate any advice.
For context, my project is looking at the potential impact of heavy metal lead (pb) on inflammasome activation in BMDMs. Right now I'm testing Pb as a secondary signal and it is dissolved in water, so water is my vehicle control.
The BMDMs I used are harvested from 8-12 weeks b6 mice and cultured with mcsf for a week before replating. I've been testing the following conditions:
- Medium alone(negative control)
- Pam alone(negative control)
- ATP alone (negative control)
- Pam + ATP (positive control)
- Pam + ATP + vehicle control
BMDMs are primed with Pam (4ug/ml) for 18 hours followed by ATP/vehicle control water for 30 mins. I then use IL-1b ELISA as a read out.
So far negative controls are good. But the main issue is the Pam+ ATP and PAm+ATP+vehicle readouts have not been consistent. The trends are not consistent between experiments. I have troubleshooted the ELISA + BMDM harvest & isolation phase to ensure the error is not there. ELISA standard curves(R2>0.98) are good and raw ODs for the replicates are almost identical within each condition. I also screen my BMDMs using cd11b FACS to ensure macrophage purity is good (>98%). So far for the Tissue culture portion, I have made sure:
- all reagents are fresh and filtered/avoid freeze thaw
- BMDMs are not dying (validated through LDH cytotoxic assay & trypan blue)
- all priming and activating reagents are made into a master mix first rather than adding direclty to the well.
- good TC practice of changing tips between every well.
If anyone has any ideas... I would greatly appreciate your help. I can't even begin testing my lead until my positive & vehicle controls are consistent :(
Thank you!
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