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FACS - Fluorescence Activated Cell Sorting - Steffen Schmitt (DKFZ) 

iBiology Techniques
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Dr. Steffen Schmitt explains the principles of FACS and describes the basic components of a droplet cell sorter. He gives advice on optimizing the yield, purity, recovery time, and viability of isolated cells.
FACS (fluorescence activated cell sorting) differs from conventional flow cytometry in that it allows for the physical separation, and subsequent collection, of single cells or cell populations. FACS is useful for applications such as establishing cell lines carrying a transgene, enriching for cells in a specific cell cycle phase, or studying the transcriptome or genome or proteome of a whole population on a single cell level. Dr. Steffen Schmitt explains the components and basic function of droplet-based cell sorters. He also provides strategies to optimize the key values in cell sorting (e.g. yield, purity, recovery time, and viability) depending on the downstream assay to be performed on the isolated cells.
Speaker Biography:
Dr. Steffen Schmitt studied biology at the Ruprecht-Karls-University of Heidelberg and completed his PhD in the Department of Cellular Immunology at the German Cancer Research Center (DKFZ). After a short post-doc, he established and led a flow cytometry core lab at the Natural Science and Medical Research Center (NMFZ) at Johannes Gutenberg University of Mainz. Since 2007, Schmitt has been head of the Flow Cytometry Unit of the Imaging and Cytometry Core Facility at the DKFZ in Heidelberg.

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29 авг 2024

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Комментарии : 11   
@TingleMingle42
@TingleMingle42 Год назад
Nice introduction to FACS! Greatly presented, Steffen, thank you!
@dmajumdar2059
@dmajumdar2059 5 лет назад
Loved the video. Thanks for the upload!
@anonviewerciv
@anonviewerciv 4 года назад
Very focused on optimization. (14:14) 3:20 Apparatus.
@PaUu912806
@PaUu912806 5 лет назад
Great video!!! Thank you!
@The9988111309
@The9988111309 4 года назад
I am still bit confused about charging the drops which we want to sort. We need to charge them at the break-off point but in the picture it shows that the flow stream is charged which means all type of cells will be charged. It makes sense to charge them according to the scattering spectrum observed, in that way we know which to sort or not. The charging is programmed according to scattered pulse values we want. Please do elaborate this otherwise I will pull all my remaining hair out. Regards, Akshit
@steffenschmitt9099
@steffenschmitt9099 4 года назад
In a cell sorter, particles are delivered in a buffered salt solution, capable conducting electrical current. We decide (based on the pattern of scatter and fluorescent signals) at the laser interrogation, if we want to sort a particle or not. We have no a bit of time to execute this sort decision, because the particle needs to travel from the laser interrogation to the droplet break off point. Once arrived there, we are charging the whole stream (here you´re right) and the leaving drop is carrying this charge and can be deflected. This works, because electrical current is travelling fast and is arriving at the same time at the break off point as the particle. (PS: Watch the slide around minute 10 “Defining drop delay - Accu Drop”: In the bottom of the screenshot you see numbers with sliders for 2nd, 3rd and 4th drop. This is correcting for potential remaining charges on the stream, once a drop has left. It ensures, that the next drop has a net-charge of zero before it can be charged again for the next sort decision.)
@tedarcher9120
@tedarcher9120 4 года назад
first we decide if we want to sort this particle and in which bucket, then we apply positive or negative charge accordingly to the whole stream, then we wait for the droplet to break off, and then we ground the stream so it loses all charge and becomes neutral again until new sort
@zerdofish9989
@zerdofish9989 2 месяца назад
FACS sorting hehe
@CiroSantilli
@CiroSantilli 5 лет назад
I wonder how this setup compares to Sphere Fluidics: ru-vid.com/video/%D0%B2%D0%B8%D0%B4%D0%B5%D0%BE-AajNacI_HEo.html
@Swervizampp
@Swervizampp 4 дня назад
Cknc
@rafikmamnya3143
@rafikmamnya3143 Год назад
Tfc nghkn😂😂😂😂
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