SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
RNA-seq from single cells? irit Sample Prep / Library Generation 282 03-04-2020 02:44 AM
Help picking up an abandoned sequencing project giror Genomic Resequencing 3 06-17-2011 10:55 AM
FACS sorting chromosomes? k-gun12 General 0 03-19-2011 07:48 AM
In Sequence: Researchers Use SOLiD to Study Gene Expression in Single Mouse ES Cells Newsbot! SOLiD 0 11-04-2008 01:00 PM

Reply
 
Thread Tools
Old 03-30-2012, 08:46 AM   #1
Simone78
Senior Member
 
Location: Basel (Switzerland)

Join Date: Oct 2010
Posts: 206
Smile Picking single cells with CellSorter (FACS in a petridish) machine?

Dear all,
we are interested in picking single cells for RNA-seq and we are looking around for semi- or fully automatic devices capable of doing that with high throughput in a very short time. Looking of the solutions currently available we came across a company commercializing a CellSorter - FACS in a petri dish. Both fluorescent and unlabelled live cells in a Petri dish observed with a microscope can be automatically recognized by computer vision and picked up by a computer-controlled micropipette fixed to the objective lens. The maximum number of cells picked up from a Petri dish was about 1,000 limited by their current sorting speed of 1 cell per second.
The company is based in Budapest (Hungary) but has no distributors in my country (Sweden). I was wondering whether anyone had heard of or worked with this machine. Is it really as easy as they claim?
Thanks a lot!
Simone78 is offline   Reply With Quote
Old 04-01-2012, 08:53 PM   #2
Jon_Keats
Senior Member
 
Location: Phoenix, AZ

Join Date: Mar 2010
Posts: 279
Default

Why not just use a classic cell sorter? Good chance you have a FACS sorter around
Jon_Keats is offline   Reply With Quote
Old 04-01-2012, 11:41 PM   #3
Simone78
Senior Member
 
Location: Basel (Switzerland)

Join Date: Oct 2010
Posts: 206
Default

Iīm not very familiar with FACS but, as far as I know, itīs very difficult or impossible to get a population of cells that is 100% pure in a short time (minutes). Ideally, we want to have 1 cell/well of a 96- or 384-well plate. This CellSorter seems to be able to do so, but I couldnīt find any paper or lab that is currently using it, and I was wondering whether itīs because itīs new or because it doesnīt work as they claim.
Simone78 is offline   Reply With Quote
Old 04-02-2012, 12:22 PM   #4
Jon_Keats
Senior Member
 
Location: Phoenix, AZ

Join Date: Mar 2010
Posts: 279
Default

Almost all current generation cell sorts, such as a BD FACS Aria, will sort single cells or multiple cells into 96 well plates. The speed is dependent on you situation but from a culture of healthy cells when you sort viable cells it should take several seconds. Find your local cell sorter this is every day kinda stuff.
Jon_Keats is offline   Reply With Quote
Old 04-03-2012, 12:28 AM   #5
Simone78
Senior Member
 
Location: Basel (Switzerland)

Join Date: Oct 2010
Posts: 206
Default

thanks a lot, Iīll take a look at it! I was just concerned about losing too many cells in the sorting process. This could be an issue when dealing with rare (and small!) samples. But the machine looks promising, albeit much more expensive than the CellSorter I was talking about , which costs only 12,000 EUR (too good to be true...)
Simone78 is offline   Reply With Quote
Old 04-10-2012, 10:08 AM   #6
454newbie
Member
 
Location: California

Join Date: Jun 2009
Posts: 17
Default

Can you post a link to the supplier? sounds interesting!
454newbie is offline   Reply With Quote
Old 04-10-2012, 01:02 PM   #7
NextGenSeq
Senior Member
 
Location: USA

Join Date: Apr 2009
Posts: 482
Default

FACS sorted cells VERY often yield degraded RNA.
NextGenSeq is offline   Reply With Quote
Old 04-11-2012, 10:31 PM   #8
Jon_Keats
Senior Member
 
Location: Phoenix, AZ

Join Date: Mar 2010
Posts: 279
Default

Quote:
Originally Posted by NextGenSeq View Post
FACS sorted cells VERY often yield degraded RNA.
Can't say that is my experience, but methods can very dramatically
Jon_Keats is offline   Reply With Quote
Old 04-13-2012, 02:12 AM   #9
Simone78
Senior Member
 
Location: Basel (Switzerland)

Join Date: Oct 2010
Posts: 206
Default

Hereīs the link of the cell picker I was talking about:
http://www.facsinapetri.com/
Simone78 is offline   Reply With Quote
Old 01-04-2013, 02:58 AM   #10
larsseq
Junior Member
 
Location: Germany

Join Date: Jan 2013
Posts: 1
Default

Hi Simone!

Have you tested the "FACS in a Petri" by now?

Would be an interesting solution for me as I want to sort fixed cells (immunofluorescence) based on image properties, so conventional FACS is not an option. However, as you mentioned, the FACS in a Petri thing seems a bit "too good to be true".

Cheers, Lars
larsseq is offline   Reply With Quote
Old 01-05-2013, 02:33 AM   #11
Simone78
Senior Member
 
Location: Basel (Switzerland)

Join Date: Oct 2010
Posts: 206
Default

we saw the cell picker at work in their lab in Budapest last spring and it seemed to be working ok (even if they had some issues with the settings while we were there). The problem is that the cell is picked in 5 ul buffer which, for our application, is way too much (we want it in 1 ul). After further optimization, last month they told us they managed to go down to 1 ul and they are soon going to send us some picked cells. We will check the integrity, try to make RNA-seq libraries out of them and then decide whether to buy it or not.
Best,
Simone
Simone78 is offline   Reply With Quote
Old 01-28-2013, 07:22 AM   #12
kwaraska
Senior Member
 
Location: Boston,MA

Join Date: Nov 2008
Posts: 122
Default

I just attended a seminar by Fluidigm. They have a plate that will select one cell into each well by "blocking" the used sites.

It takes a specific machine but from there you can stain them, isolate RNA even create NGS RNA-seq libraries.

Check that out-might be easier than a FACS-epecially if you don't have one!
kwaraska is offline   Reply With Quote
Old 01-28-2013, 11:06 PM   #13
Simone78
Senior Member
 
Location: Basel (Switzerland)

Join Date: Oct 2010
Posts: 206
Default

yes, the C1 single-cell auto prep system from Fluidigm seems to be a great instrument. The problem I see is that you need to buy an expensive machine + their reagents (probably very expensive as well). We finally bought a cell picker from CellSorter (http://facsinapetri.com/), expected delivery in 2 months. Iīll post my results and impressions about that.
Simone78 is offline   Reply With Quote
Old 03-18-2014, 07:32 AM   #14
Jaykay
Junior Member
 
Location: Germany

Join Date: Mar 2014
Posts: 1
Default

Hi Simone,
just wondered if you already got some results with the CellSorter? I want to sort some large macrophages for RNA analysis, which would not survive being sorted by a FACS sorter.
Jaykay is offline   Reply With Quote
Old 03-19-2014, 08:45 AM   #15
Simone78
Senior Member
 
Location: Basel (Switzerland)

Join Date: Oct 2010
Posts: 206
Default

In principle it works, but there is a big "BUT". There are a few bugs in the software. Sometimes we are ready to pick cells but we have to restart the program because itīs not responding. This increases the time the cells are on the Petri dish, which increases stress (thus, decreasing viability) and lead to unwanted changes in the expression profile. This, at least, itīs my interpretation of things. To give you an idea of what I mean, I tell you the conclusion of our latest experiment. We compared single nuclei (GFP labelled) picked with CellSorter or by FACS. We got comparable libraries before seq (similar yield and avg size of library), but they cluster apart from each other...the FACS-sorted nuclei on one side of the plot, the CellSorter ones on the other! Donīt know exactly what that means. Again, it might be an artificial change in the expression profile due to the longer time cells were on the petri dish. Or it might be true (?).
With CellSorter there are also issues related to how strongly the cells are attaching to the dish. They must be loosely attached in order to be able to pick them, but if they attach too much you wonīt be able to get them into the capillary. Of course, the longer it takes for the picking the higher the number of cells that are missed. For some cells, even adding Tryple Express (mild tripsinization) doesnīt help.
In conclusion, I am not sure this is the best system but it seems to be better than other micromanipulators and itīs definitely cheaper. I am still skeptic it is worth the money we paid, though...
Best,
Simone
Simone78 is offline   Reply With Quote
Old 05-16-2014, 11:09 PM   #16
Loligo
Junior Member
 
Location: Berlin

Join Date: May 2014
Posts: 1
Thumbs up Cellsorter

I am using one for single cell rna seq. Best thing to get all dif. Cells of a heterogenic tissue in one prep. Easy to use and less stress for the cells than a facs. No celltype failed using the picker.
Loligo is offline   Reply With Quote
Old 06-25-2014, 11:27 PM   #17
MarkM
Junior Member
 
Location: Germany

Join Date: Jun 2014
Posts: 1
Default

Hi Simone,
we are thinking about buying one of that Cellpickers to pick single cells and we are as well not sure if it is worth it.
Are there some more news about that or further experience you had with the device ?
If you are interested may be it is possible to have a personal discussion on that ?
Regards
Mark
MarkM is offline   Reply With Quote
Old 06-28-2014, 05:14 AM   #18
Simone78
Senior Member
 
Location: Basel (Switzerland)

Join Date: Oct 2010
Posts: 206
Default

Hi!
I didnīt have a chance to pick more cells with the cell picker. Iīm usually using FACS or I am picking them manually (if I need just a few). However, a colleague of mine is using it and he gets good results. I tried to send you a private mail but your account settings donīt allow me to do that. If you change your settings I can send you his contact details.

Best,
Simone
Simone78 is offline   Reply With Quote
Old 11-13-2014, 01:01 AM   #19
eab
Member
 
Location: Maryland

Join Date: May 2011
Posts: 66
Default

Has anyone had any further progress with this device?
eab is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 03:18 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO