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Category 'Antibodies'
Status: Answered
Views: 600

8-Antibody-Panel validation

Bogdan Manescu (Author)

Saturday, February 17, 2018 12:03 AM

Hi there, this is my first post.

My question is about an antibody panel for one of our upcoming projects. The panel is quite complex and is comprised of 8 fluorochromes.

Our technician worked on this panel and sent it to an *unnamed* provider for validation. It's supposed they have all the resources to check and validate such a panel. To our surprise, they sent back another panel (you'll see it in the images) which seems ok with ONE exception: although excited by different lasers, APC-Cy7 and PE-Cy7 emit at the same wavelength

As far as i know, all 3 lasers come in the same beam, meaning i will get both fluorochromes excited at the same time, then how can the cytometer tell which signal is whom since it can't do a sequential analysis? Now maybe there's something i don't know and they do. I use a FACSAria III cytometer.

I'd greatly appreciate any help!

Link for the panel overview
https://imgur.com/a/KvCWk

Silvestro Volpe

Saturday, February 17, 2018 9:17 AM

Time delay....

 

John Stokes

Best answer
Saturday, February 17, 2018 11:58 AM

Hi Bogdan, I believer the FACSAria III has a parallel laser arrangement rather than collinear. Therefore, the lasers are not actually on the same beam, and will not excite the fluorophores at the same time.  As Silvestro indicated, the signals will be "stitched" together later in the delay electronics.

Silvestro Volpe

Saturday, February 17, 2018 4:27 PM

Very well John. Very exhaustive.

 

Roopesh Singh

Saturday, February 17, 2018 11:24 PM

Hi 

Bogdan Manescu

I will add some more information to the John's answer. 

Each laser has its own filters for detectiong emmision spectrum. That means the fluorochromes PE-cy7 and APC-cy7 though emit in the same range in 780/60, but since they excited essentially by different lasers, the emmision spectra will not interfer each other. You can safely use them in your panel. I am using both of these in FACS ARIA II and LSR II. 

I think ARIA II has similar laser arrangement.

good luck.

Bogdan Manescu (Author)

Sunday, February 18, 2018 9:49 AM

Hi there again, thank you very much for your replies. You are all right about the time delay and it's my fault for not reading the User Manual carefuly. After posting here, later that night, i actually got to check the FACSAria III User Manual and i found the answer ( https://imgur.com/a/6Vqxn ).

Just for the record, i am just a 5th year student of Medicine and have recently had my first contact with the citometry community and i'm still learning. That explains such a laughable error on my side :D

I will probaly come back here repeatedly since i'm still learning the whole citometry stuff. Thank you again for your answers! :)

Bogdan Manescu (Author)

Sunday, February 18, 2018 9:52 AM

Although all of you provided the correct answer, i'll give the star to John Stokes as he was the first one to get to the details.

Rachel Gerstein

Sunday, February 18, 2018 12:40 PM

as a "new to cytometry" person, you should know how to test whether reagents interfer or not, as minimizing compensation required can help in panel design.  for your case, stain cells with your APC-Cy7 and your PE-cy7 antibodies, and try to set this up so that you have a good number of both negative and positive.  collect each and then plot each vs the "other" color.

you could post the results here or email me, whichever you prefer