Membrane Stability under various RTP Treatments: Retry
Previously W514 was tested with different RTP conditions (varying time at 800C, all in Ar); the results were difficult to interpret as the cells were confluent (in some cases it was hard to see membrane breakage). We therefore decided to run a similar test again, with a slight change in O2 addition during RTP. O2 was only run for a short time (30 seconds) as Ar was ambient for duration of RTP treatment; in contrast the condition in the W514 test where only O2 was ran. The conditions run for discoloration without cells (EGM +10% FBS) and membrane stability with cells (HUVEC, P6) were:
- no treatment
- RTP in Ar @ 800C for 1 Minute
- RTP in Ar @ 800C for 5 Minutes
- RTP in Ar @ 800C for 10 Minutes
- RTP in Ar @ 800C for 5 Minutes with a “puff” of O2 for 30 second
Results of discoloration show a similar trend as observed in the W514, with RTP discoloration for all time points starting around the same time. Furthermore it does not appear that O2 in RTP prevented discoloration. Lastly, Chris mentioned that RTPed samples discolor non-uniformly; this happened in this study, suggesting non-uniform RTP processing, handling, or other unknown factors.
For membrane stability samples, HUVEC were seeded at a low confluence with membranes monitored over time, using the bead assay when membrane condition was questionable. Failure of untreated samples occurred completed by 23 hours, as expected by the complete discoloration seen in 23 hours. The RTP with a puff of O2 sample broke by 44 hours; only 1 of 11 membranes survived gentle handling to set up the experiment. Failure of RTP for 1, 5, and 10 minutes in Ar started within 44 hours, with complete failure of all samples by 91 hours. Samples that were RTPed for 5 minutes stayed intact for the longest time.
In conclusion, RTP with O2 does not seem useful, as it does not dramatically improve membrane stability and these samples are very difficult to handle. It also appears that varying RTP time between 5 and 10 minutes does not seem to have a significant effect on membrane stability. It may be the case that even longer RTP times would improve stability, but Dave said the RTP draws so much current that it is unrealistic to do so.
Interesting. Have we tried RTP for 5 min in Ar FOLLOWED by a minute or two in the O2? The Ar anneal may be some type of reorganization, that may still benefit from some O2.