Feb Interlab samples

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Nicholas DeLateur

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Feb 21, 2017, 1:29:48 PM2/21/17
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Hey everyone,

I grew up the samples last night and prepared them. 
Today I gave them to my lab manager to ship and she accidentally sent all three to John Sexton, rather 1 to John Sexton, 1 to Ariel Hecht, 1 to David Ross. The tracking number is Tracking # 778474939276.

I apologize for the inconvenience. 

If John and Sebastian can easily and quickly turn them around, we'll do that. 

If not, I'll grow up another batch and we'll do another run in the near future. 

Yours, 
Nicholas 
 

Nicholas DeLateur

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Feb 21, 2017, 1:33:32 PM2/21/17
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Nevermind. I have been informed that it might be salvaged, the lab manager intercepted the package and will open, divide into 3, and re-attempt. 

At the very least they're all set up and on dry-ice...

Please stand by until I have a better understanding
And no I don't know what this tracking number is for then. 

Yours in confusion and frustration,
Nicholas

Nicholas DeLateur

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Feb 21, 2017, 2:15:31 PM2/21/17
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So while the package was ordered it was not yet picked up and thus salvaged. 

Below are the putative tracking numbers
John  778474939276
Ariel 778477528932
David 778477591577

John Sexton

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Feb 21, 2017, 3:24:56 PM2/21/17
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Awesome! Thanks Nicholas. Looking forward to the data this will yield!

-John

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Jake Beal

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Feb 21, 2017, 3:41:45 PM2/21/17
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What a mess! Thank you so much, Nicholas!

Thanks,
-Jake
(sent from my phone)

-John

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John Sexton

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Feb 22, 2017, 12:18:08 PM2/22/17
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Samples received in HTX!

Nicholas, can you elaborate on what the "1" through "5" tube labels mean? Thanks.

-John

-John

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Nicholas DeLateur

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Feb 22, 2017, 12:21:32 PM2/22/17
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Sorry, my bad, I thought I sent that out. 
TUBE 1: pL1f1EV
TUBE 2: pL1f1362
TUBE 3: pL1f1357
TUBE 4: pL1f1372
TUBE 5: pL1f1285
I'll update the experimental document. 

-John


-John

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Ariel Haim Hecht

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Feb 22, 2017, 1:58:34 PM2/22/17
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Package received at Stanford 

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Nicholas DeLateur

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Feb 22, 2017, 3:04:24 PM2/22/17
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I did my voltage testing today. 

Tube 2 containing strain sFW006 behaved poorly with low counts and a very broad peak (I don't think we're hitting the gold standard on this one), but the other samples are all behaving well in my hands. Sorry about that. 

Nicholas 

Package received at Stanford 

-John


-John

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John Sexton

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Feb 22, 2017, 3:45:02 PM2/22/17
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I can corroborate this.

Tube 2 containing sFW06 had almost no discernible cell events. There were a few uncommonly green-fluorescent events, which I attempted to use to determine a low, medium, and high GFP voltage, but I don't trust these events because they were simultaneously nearly as fluorescent as the sFW08 strain in the mCherry channel.

Nicholas, if you saw discernible cell events from the medium GFP sample (sFW07), perhaps we should ditch sFW08 and determine GFP voltages based on sFW07? (I haven't looked at an sFW07 sample yet).

I was able to identify a peak for the sFW08 sample, but it had a long tail (it did not look log-normally distributed). And while the event rate for that PBS-diluted biological sample was nonzero, I felt like it could be increased (i.e. increase the concentration of the initial shipped biological sample if this ever gets done again).

I also saw a lot of non-fluorescent events in roughly the same FSC-SSC region as the cell events I was detecting. I went ahead and measured both sFW06 and sFW08 samples along with my 0.2um filtered-PBS sample in case we want to explore that more. A PDF of my notes and my data files are in "2017_Feb_interlab/data/Rice/VoltageDetermination".

-John

Package received at Stanford 

-John


-John

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Ross, David J. (Fed)

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Feb 22, 2017, 3:46:29 PM2/22/17
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Samples received at NIST Gaithersburg.

Package received at Stanford 

 

-John

 

-John

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John Sexton

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Feb 22, 2017, 3:52:05 PM2/22/17
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Nicholas, if you saw discernible cell events from the medium GFP sample (sFW07), perhaps we should ditch sFW08 and determine GFP voltages based on sFW07? (I haven't looked at an sFW07 sample yet).

Ahhh sorry I got my strain labels confused. Should say: Ditch sFW06* (strong GFP) in favor of sFW07 (medium GFP)

Package received at Stanford 

 

-John

 

-John

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Nicholas DeLateur

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Feb 22, 2017, 3:54:02 PM2/22/17
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This is what I got with 10,000 raw events gated for E. coli and bead-scaled
Inline image 1Inline image 2


Package received at Stanford 

 

-John

 

-John

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Nicholas DeLateur

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Feb 22, 2017, 3:55:55 PM2/22/17
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I think that's entirely viable, although if you use Medium Green to set your Green Voltage, I'd say shoot for setting it where you'd still expect it to be. It was chosen because it's about ~10 fold less bright. So if you were planning on setting your voltages to put sFW06 at 10**5, 10**4, 10**3, simply instead use sFW07 to set the voltages such that sFW07 is at 10**4, 10**3, 10**2. 

Package received at Stanford 

 

-John

 

-John

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Ariel Haim Hecht

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Feb 23, 2017, 3:26:02 PM2/23/17
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I just ran my voltage test and also saw very few cell events in the strong green sample. i was able to get enough signal to ballpark the voltages, and checked them against the medium green and saw that they were roughly 10-fold apart, and the medium green was roughly 10-fold below the strong green, so that looks good to me.

Dave—I wanted to cross-check voltages with you, to see if we’re using the same settings. I’ve got:

FSC-350
SSC-350
YL2-310, 400, 550
BL1-200, 280, 375

Thresholds: FSC 200, SSC 2000.

-Ariel

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Ross, David J. (Fed)

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Feb 23, 2017, 4:20:24 PM2/23/17
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I just finished the perf. test and am about to start the voltage setting. stay tuned…

 

From: sbsc-flow...@googlegroups.com [mailto:sbsc-flow...@googlegroups.com] On Behalf Of Ariel Haim Hecht
Sent: Thursday, February 23, 2017 3:26 PM
To: sbsc-flow...@googlegroups.com
Subject: Re: [sbsc-fc] Feb Interlab samples

 

I just ran my voltage test and also saw very few cell events in the strong green sample. i was able to get enough signal to ballpark the voltages, and checked them against the medium green and saw that they were roughly 10-fold apart, and the medium green was roughly 10-fold below the strong green, so that looks good to me.

Package received at Stanford 

 

-John

 

-John

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Ross, David J. (Fed)

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Feb 23, 2017, 5:07:50 PM2/23/17
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NIST Gaithersburg voltages:

FSC: 350

SSC: 450 (I had to bump this up relative to Ariel’s to avoid losing the sub-population marked on the attached plot)

YL2: 290, 375, 500

BL1: 210, 275, 350

Scatter plot.2.bmp

Ariel Haim Hecht

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Feb 23, 2017, 5:51:32 PM2/23/17
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Cool, that sounds pretty close, certainly on BL1/YL2.

Package received at Stanford 
 
-John

 


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<Scatter plot.2.bmp>

John Sexton

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Feb 23, 2017, 7:23:10 PM2/23/17
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Rep 1 for Rice is uploaded. It took me ~2 hours to prepare samples and measure.

I didn't see many cells with sFW05 (blank) or sFW06 (strong GFP) again. I'm definitely feeling like future iterations should standardize the cell mass that gets distributed.

-John

Ross, David J. (Fed)

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Feb 24, 2017, 9:31:34 AM2/24/17
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NIST Gaithersburg data is uploaded.

 

We ran the beads twice: once with the SSC and FSC voltages the same as for the E. coli, and once with the SSC voltage adjusted to center the beads. See the notes document for details.

Ross, David J. (Fed)

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Feb 24, 2017, 9:38:20 AM2/24/17
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My plan for the next two replicates is to only do the beads with re-centering (i.e. adjusting the SSC voltage differently for E. coli and for beads).

 

If anyone wants me to do something different please let me know by 3pm today (Eastern time).

Ariel Haim Hecht

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Feb 24, 2017, 5:22:25 PM2/24/17
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NIST-Stanford data collection and upload for all 3 replicates is complete.

John Sexton

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Feb 24, 2017, 9:18:46 PM2/24/17
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Rep 2 and Rep 3 for Rice are uploaded.

-John

Nicholas DeLateur

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Feb 26, 2017, 5:40:20 PM2/26/17
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MIT data is uploaded.

-John

-John

-John

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