Initial Freeze Thaw Procedure Validation

[Nicholas DeLateur]

There is a new folder in the interlab folder called "InitialFreezeThawProcedureValidation". It contains all raw data, analyzed data, and methods. 

Here is the ENTIRETY of the procedure for the end-user:

Retrieve 1 Eppie tube of each strain from the -80

Immediately place in 42 degree water bath for exactly 60 seconds

Add 990 uL of 1X PBS

Briefly vortex

Transfer 200 uL to plate

Perform Flow Cytometry

Here are the results:

I ran it with mCherry strains because that's what I had lying around at the moment. 

It is my understanding that we should use GFP since that is the common channel we all have, and/or we understand the beads at a GFP context more than other contexts. This is a trivial change on my end and I have already secured the plasmids with GFP instead of mCherry. 

Shipping samples on dry ice isn't particularly trivial though. I'll set up a meeting with my EHS representative to make sure I do it as well as possible. 

The protocol was designed and tested such that the cells can be stored without the need for immediate measurement. If you wait a day or two from receiving them t should be fine, but let's still try to coordinate and plan to make sure people receive them right before or close to when they plan to measure. 

Because I'm not particularly smart, I don't have everyone's shipping address in one central location and gmail-search-fu isn't quite strong enough. I will reach out to everyone again individually and then put it in the Interlab folder for when I forget again very soon. My apologies.

Happy to answer any questions,

Nicholas  

[Jake Beal]

Hi, Nick:

This looks very nice and about as tight as I could expect to see from the instrument --- great!

Looking at a few of the individual files: their distributions also look nice and tight, which is excellent.

Now, before we go the next steps and talk about shipping, I'd like to make sure we've all chewed over the experimental plans carefully so that we can make sure everybody's efforts are well used and this goes right the first time. I've coped the protocol statement that you put up top into the "Driving scenarios ..." file; can you also supply information on what samples you've got available, and then we can all comment, question, and clarify?

Thanks,

-Jake

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[Ariel Haim Hecht]

Hi Nick,

These data look great! And thanks for spelling out the protocol!

I have a question about the difference between mean and geometric mean of the two samples, which I ask only because I’m not very familiar with analyzing flow cytometry data. How would someone make the choice to use mean or geometric mean when analyzing the data? 

Thanks!

Ariel

On Jan 24, 2017, at 7:21 PM, Nicholas DeLateur <nichola...@gmail.com> wrote:

There is a new folder in the interlab folder called "InitialFreezeThawProcedureValidation". It contains all raw data, analyzed data, and methods. 

Here is the ENTIRETY of the procedure for the end-user:

Retrieve 1 Eppie tube of each strain from the -80

Immediately place in 42 degree water bath for exactly 60 seconds

Add 990 uL of 1X PBS

Briefly vortex

Transfer 200 uL to plate

Perform Flow Cytometry

Here are the results:

<image.png>

I ran it with mCherry strains because that's what I had lying around at the moment. 

It is my understanding that we should use GFP since that is the common channel we all have, and/or we understand the beads at a GFP context more than other contexts. This is a trivial change on my end and I have already secured the plasmids with GFP instead of mCherry. 

Shipping samples on dry ice isn't particularly trivial though. I'll set up a meeting with my EHS representative to make sure I do it as well as possible. 

The protocol was designed and tested such that the cells can be stored without the need for immediate measurement. If you wait a day or two from receiving them t should be fine, but let's still try to coordinate and plan to make sure people receive them right before or close to when they plan to measure. 

Because I'm not particularly smart, I don't have everyone's shipping address in one central location and gmail-search-fu isn't quite strong enough. I will reach out to everyone again individually and then put it in the Interlab folder for when I forget again very soon. My apologies.

Happy to answer any questions,

Nicholas  

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[Jake Beal]

On Wed, Jan 25, 2017 at 3:44 PM, Ariel Haim Hecht <arih...@stanford.edu> wrote:

I have a question about the difference between mean and geometric mean of the two samples, which I ask only because I’m not very familiar with analyzing flow cytometry data. How would someone make the choice to use mean or geometric mean when analyzing the data? 

There are typically two dominant components of variation in flow cytometry measurements:

- cell-to-cell variation which is typically log-normally distributed

- instrument noise, which is typically normally distributed

Thus, if you are dealing with strong fluorescence, geometric statistics represent the distribution better, but if you are dealing with fluorescence near zero, arithmetic statistics represent the distribution better.  In between (around 10^1 - 10^2 a.u.) it's problematic, which is what the "logicle" (bi-exponential) plots are used for.  I'm very uncomfortable with them, however, because their statistical meaning is unclear and they are often used incorrectly.

This is the reason that I have recommended we take measurements at 10^3 - 10^5 a.u.

Thanks,

-Jake

[Ariel Haim Hecht]

I see, thanks! Yes, seems like operating in a regime where biological variability dominates makes sense. 

-Ariel

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[Nicholas DeLateur]

These samples were pL1f1-pLacIq-RBS[variable]-mCherry-Terminator. Where [variable] is 30, 31, 32, and 33. 

I have pL1f1-pLacIq-RBS[variable]-GFP-Terminator, where [variable] is 30, 31, 32, and 33.

Bringing out the scope a bit more, I have 2 different promoters and 17 different RBS. 

Does that help answer your question about samples? 

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[Nicholas DeLateur]

I don't know, hence why I did both, and am eminently interested in learning more how to better make that choice and defer to Jake and Brian and the others on this, and of course thanks to Jake for his quick summation below as well. 

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