NIST Stanford data collected, will be uploaded shortly

[Ariel Haim Hecht]

Hi all,

I got the data collected today. To me it looked mostly good, with a few key details:

1) Strain 93 appears to have lost its ability to generate red fluorescence. I saw great red signal from 90, great green signal from 91, but strain 93 showed only green. There might be a touch of red buried near the noise, but on first glance did not appear to be any red. My first guess would be some sort of mutation in the mCherry coding region (paging Dave — evolutionary stability).

2) It took about 2.5 hours to collect data at all 3 voltages. I’m assuming that since the cells were bathing in kanamycin, probably not a huge deal, but just wanted to note it. This was mostly due to me still figuring out how to optimize use of the autosampler. I collected the first voltage at 12.5 uL/min, 1e5 events per sample. For the second two I upped it to 25 uL/min, 2.5e4 events per sample to speed things up.

3) There was significant carry-over between the wells using the autosampler. I ran a blank solution of PBS between each sample, plus two Attune rinse cycles between each sample. Not sure if this is a machine-specific issue, but probably something for at least Dave and I to keep an eye on.

Data will be uploaded shortly.

-Ariel

[Jake Beal]

Thank you, Ariel; I will look at the data once you have uploaded it.

With regards to issue #1: this is another confirmation of my concerns about use of calibrants that require culturing. Notice that we are immediately deep into speculation about extremely complex biological mechanisms and processes, with no straightforward way of diagnosing possible problems.

Along those lines, I hope to hear an update on frozen sample shipping from Nick and Brian on Monday...

Thanks,

-Jake

-Ariel

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[Ariel Haim Hecht]

Thanks Jake. The data is up and ready to roll. I’ve included a readme sheet in the folder in case the file namings are unclear.

I agree that this highlights the challenges with using cultured cells to compare measurements across sites. While using frozen cells mitigates some of these issues, I’m not sure that it fully eliminates them. I guess we’ll find out!

Along those lines, what will the frozen cells be suspended in? Glycerol? I’m sure we’ll be diluting the cells in PBS, but do we know if there is an amount of glycerol that is safe for the cytometers? I’d hate to get our fluidics gunked up.

-Ariel

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