Follow-on from today's meeting

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Jake Beal

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Jan 30, 2017, 5:27:03 PM1/30/17
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Everybody who will be involved in the experiment, please review it in detail. When you are satisfied, write and say you are ready to perform. When everybody confirms, we will proceed to coordinate calendars for shipments.

Thanks,
-Jake

Ariel Haim Hecht

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Feb 2, 2017, 2:02:42 PM2/2/17
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I am ready to perform.

-Ariel

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Ross, David J. (Fed)

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Feb 2, 2017, 3:39:10 PM2/2/17
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I made a few small changes:

The PBS should be filtered even if it is freshly made.

I reduced the collection volume to 160 uL instead of 175 uL. I double checked, and the dead volume on the Attune is 30 uL, so acquiring 160 is close to the max – and that leaves a bit in the well to make sure we don’t pull in bubbles.

 

If everyone else agrees with these changes, we are ready to perform.

 

Also, next Monday, I will be at a conference, so I will miss the phone Google Hangout-

 

Thanks-

Jake Beal

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Feb 2, 2017, 3:45:41 PM2/2/17
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That all looks fine to me.

Thanks,
-Jake


On Thu, Feb 2, 2017 at 2:39 PM, Ross, David J. (Fed) <david...@nist.gov> wrote:

I made a few small changes:

The PBS should be filtered even if it is freshly made.

I reduced the collection volume to 160 uL instead of 175 uL. I double checked, and the dead volume on the Attune is 30 uL, so acquiring 160 is close to the max – and that leaves a bit in the well to make sure we don’t pull in bubbles.

 

If everyone else agrees with these changes, we are ready to perform.

 

Also, next Monday, I will be at a conference, so I will miss the phone Google Hangout-

 

Thanks-

 

 

 

From: sbsc-flow-cytometry@googlegroups.com [mailto:sbsc-flow-cytometry@googlegroups.com] On Behalf Of Ariel Haim Hecht
Sent: Thursday, February 02, 2017 2:03 PM
To: sbsc-flow-cytometry@googlegroups.com
Subject: Re: [sbsc-fc] Follow-on from today's meeting

 

I am ready to perform.

 

-Ariel

On Jan 30, 2017, at 2:27 PM, Jake Beal <jake...@gmail.com> wrote:

 

Everybody who will be involved in the experiment, please review it in detail. When you are satisfied, write and say you are ready to perform. When everybody confirms, we will proceed to coordinate calendars for shipments.

 

Thanks,

-Jake

 

 

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Nicholas DeLateur

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Feb 3, 2017, 6:38:44 PM2/3/17
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Hi Jake.

I don't see the reason for the technical replicates at each voltage here. It's not testing much of anything having 3 wells of 200 uL of the exact same sample, much like you wouldn't test anything more having 6 wells of 100 uL each would. Collecting at different voltages seems more interesting, especially since the math doesn't really work out if you want three technical replicates of three different voltages. This would be 200x9 = 1800 uL of cells. The protocol only gives you 1000 uL of cells. If I had to choose one set of three I'd much rather then different voltages, but I'm happy to know thoughts on this. 

As far as what strains I'll be sending, I don't have a good two-colour. Getting rid of positional or resource or proximity or context effects is not something that's been overcome, and the sample I sent around last time is too strong an expressor and it results in a negative population we've seen. Instead I'll send a Strong and a Medium for GFP and a Strong and a Medium for mCherry and an Empty Vector. These 5 should be comparable and interesting and more well-behaved than my "what do I have lying around" batch last time. I will include the maps in a folder in the Interlab and a key in the experimental workflow (I promise to try my best not to mislabel one like last time). 

Also as far as the analysis goes, we all do it a bit differently with our respective programs that do roughly the same things, just wanna make sure that's clear there; for example I don't use a Gaussian mixture model but rather a combination of SSC-A, FSC-A, and FSC-H, as well as the other steps with the Cytoflow package. So the analysis is somewhat a different beast than the data collection. 

For shooting blanks between samples, this section should be variable. I need 0. Some people from my understanding need more than 1. I prefer the wording "Place as many focusing/sheath fluid blanks your machine requires before each sample. If you don’t know what this number is, alert the group ASAP and we will help you figure it out". I distinctly remember some cytometers being discussed requiring 4 or 5?

Shoot for as close as you can to 0.5 uL/second flow rate (record actual)

Sorry a bit rushed but I hope that makes sense. 

Thanks,
Nicholas 





Ross, David J. (Fed)

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Feb 3, 2017, 7:18:09 PM2/3/17
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My understanding of the protocol is that we're supposed to do one technical replicate for each voltage, and then repeat the whole process 3 different times (e.g. on three different days). So, the three wells of 200 uL each are one well for each of the three voltages. If that is not clear, then we should edit the document so that it is.


Also, what is the measured carry-over rate that you get with zero focusing fluid blanks?


Thanks-



From: sbsc-flow...@googlegroups.com <sbsc-flow...@googlegroups.com> on behalf of Nicholas DeLateur <nichola...@gmail.com>
Sent: Friday, February 3, 2017 6:38 PM
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Subject: Re: [sbsc-fc] Follow-on from today's meeting
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Jake Beal

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Feb 6, 2017, 2:05:20 PM2/6/17
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On Fri, Feb 3, 2017 at 5:38 PM, Nicholas DeLateur <nichola...@gmail.com> wrote:
Hi Jake.

I don't see the reason for the technical replicates at each voltage here. It's not testing much of anything having 3 wells of 200 uL of the exact same sample, much like you wouldn't test anything more having 6 wells of 100 uL each would. Collecting at different voltages seems more interesting, especially since the math doesn't really work out if you want three technical replicates of three different voltages. This would be 200x9 = 1800 uL of cells. The protocol only gives you 1000 uL of cells. If I had to choose one set of three I'd much rather then different voltages, but I'm happy to know thoughts on this. 

As David said, the idea is to repeat the whole process (defrost to measurement) three separate times. That will give us an understanding of how much variability is being introduced by that protocol (hopefully very little).
 
As far as what strains I'll be sending, I don't have a good two-colour. Getting rid of positional or resource or proximity or context effects is not something that's been overcome, and the sample I sent around last time is too strong an expressor and it results in a negative population we've seen. Instead I'll send a Strong and a Medium for GFP and a Strong and a Medium for mCherry and an Empty Vector. These 5 should be comparable and interesting and more well-behaved than my "what do I have lying around" batch last time. I will include the maps in a folder in the Interlab and a key in the experimental workflow (I promise to try my best not to mislabel one like last time). 

That's fine; I've updated the experiment document. Can you please respond to John's questions about the exact green and red proteins?
 
Also as far as the analysis goes, we all do it a bit differently with our respective programs that do roughly the same things, just wanna make sure that's clear there; for example I don't use a Gaussian mixture model but rather a combination of SSC-A, FSC-A, and FSC-H, as well as the other steps with the Cytoflow package. So the analysis is somewhat a different beast than the data collection. 

Yes: we'll have some back-and-forth on analysis after we've started the physical lab part.
 
For shooting blanks between samples, this section should be variable. I need 0. Some people from my understanding need more than 1. I prefer the wording "Place as many focusing/sheath fluid blanks your machine requires before each sample. If you don’t know what this number is, alert the group ASAP and we will help you figure it out". I distinctly remember some cytometers being discussed requiring 4 or 5?

Shoot for as close as you can to 0.5 uL/second flow rate (record actual)

Can you please put these as comments into the drive document for discussion?

Thanks,
-Jake

John Sexton

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Feb 9, 2017, 5:03:05 AM2/9/17
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I changed some things in the Experimental Design section in an attempt to organize/clarify it (in a manner which hopefully didn't significantly change the meanings of previous statements). I also added a bunch of comments (apologies ahead of time for being so nit-picky...). Once those are addressed, we (Rice / John+Sebastian) are on board. (I have not had a chance to review the analysis section yet, though)

-John

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Jake Beal

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Feb 9, 2017, 6:27:27 AM2/9/17
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On Thu, Feb 9, 2017 at 4:02 AM, John Sexton <john.t...@rice.edu> wrote:
I changed some things in the Experimental Design section in an attempt to organize/clarify it (in a manner which hopefully didn't significantly change the meanings of previous statements). I also added a bunch of comments (apologies ahead of time for being so nit-picky...). Once those are addressed, we (Rice / John+Sebastian) are on board. (I have not had a chance to review the analysis section yet, though)

Please do *not* apologize, John. The whole point is to make sure everybody is acting as equivalently as possible, and queries like you're making are absolutely required to ensure that.  I've resolved some and added responses to some, for your perusal. Others require input from Nicholas or other lab folks.

Thanks,
-Jake
 

Jake Beal

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Feb 9, 2017, 6:30:07 AM2/9/17
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Also, since I haven't seen email go out about it yet:

In our meeting on Monday, we proposed for the experiment to be run on the week of February 20 - 24, starting Feb 21 or 22 (depending on whether Nicholas can ship on President's day or not).  These dates are confirmed to work for MIT, NIST Stanford, and Rice.  NIST Gaithersburg, can you please confirm as well?

Also, if any lurkers on the list wish to participate, speak up and you will be welcome to join. :-)

Thanks,
-Jake

Ross, David J. (Fed)

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Feb 9, 2017, 8:30:58 AM2/9/17
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Yes. We confirm. That week works for NIST Gaithersburg.


Thanks-



From: sbsc-flow...@googlegroups.com <sbsc-flow...@googlegroups.com> on behalf of Jake Beal <jake...@gmail.com>
Sent: Thursday, February 9, 2017 6:30 AM

To: sbsc-flow...@googlegroups.com
Subject: Re: [sbsc-fc] Follow-on from today's meeting
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Jake Beal

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Feb 9, 2017, 8:36:11 AM2/9/17
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Excellent! Please everybody make sure that Nicholas has the correct shipping information to send you -80 samples.

Thanks,
-Jake


On Thu, Feb 9, 2017 at 7:30 AM, Ross, David J. (Fed) <david...@nist.gov> wrote:

Yes. We confirm. That week works for NIST Gaithersburg.


Thanks-




Sent: Thursday, February 9, 2017 6:30 AM

Subject: Re: [sbsc-fc] Follow-on from today's meeting
Also, since I haven't seen email go out about it yet:

In our meeting on Monday, we proposed for the experiment to be run on the week of February 20 - 24, starting Feb 21 or 22 (depending on whether Nicholas can ship on President's day or not).  These dates are confirmed to work for MIT, NIST Stanford, and Rice.  NIST Gaithersburg, can you please confirm as well?

Also, if any lurkers on the list wish to participate, speak up and you will be welcome to join. :-)

Thanks,
-Jake

On Thu, Feb 9, 2017 at 5:27 AM, Jake Beal <jake...@gmail.com> wrote:
On Thu, Feb 9, 2017 at 4:02 AM, John Sexton <john.t...@rice.edu> wrote:
I changed some things in the Experimental Design section in an attempt to organize/clarify it (in a manner which hopefully didn't significantly change the meanings of previous statements). I also added a bunch of comments (apologies ahead of time for being so nit-picky...). Once those are addressed, we (Rice / John+Sebastian) are on board. (I have not had a chance to review the analysis section yet, though)
Please do *not* apologize, John. The whole point is to make sure everybody is acting as equivalently as possible, and queries like you're making are absolutely required to ensure that.  I've resolved some and added responses to some, for your perusal. Others require input from Nicholas or other lab folks.

Thanks,
-Jake
 

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