02:16:52	William Telford:	Attend CYTO 2021 for free this year:  https://www.cytoconference.org
02:17:24	William Telford:	CYTO U flow cytometry education modules are currently free:  https://learning.isac-net.org/
02:19:22	REKHA GOUR:	NAMASTE TO ALL FROM INDIA
02:20:01	Pragati Pradhan:	Namaste Rekha mam from Nepal.
02:21:55	William Telford:	CYTO U:  https://learning.isac-net.org/
02:27:15	William Telford:	All workshop content at:  https://www.cytometryunderground.com/TU_Flow_2_2021.html
02:33:15	William Telford:	Please feel free to ask questions in chat!  The faculty will be monitoring.
02:35:02	Hemant Agrawal:	namaste everyone. Enjoy these wonderful lectures
02:35:23	Pragati Pradhan:	Namaste Hemant sir.
02:36:14	Madan Raj Pandey:	namaste everyone
02:36:38	William Telford:	A link to the February first workshop is on the new workshop site - also at https://www.cytometryundergorund.com/TU_Flow_2021.html
02:36:54	William Telford:	All the first workshop videos are there.
02:37:29	Madan Raj Pandey:	Thanks Bill for information.
02:38:21	Sud:	couldnot open the link
02:39:03	William Telford:	Sorry, I mis-typed - old content at https://www.cytometryunderground.com/TU_Flow_2021.html
02:39:36	Sudarshan GC:	thank you
02:39:44	Sudarshan GC:	It works
03:29:46	William Telford:	Zosia Maciorowski contact:  zosiamaciorowski@gmail.com
03:29:49	Reshma Tuladhar:	What makes stain index of one cytometer different from other cytometer for same fluorochrome?
03:31:05	William Telford:	Derek Davies contact:  Derek.Davies@crick.ac.uk
03:31:41	William Telford:	Differences in staining index between instruments are usually instrument-related - some instruments are more sensitive than others.
03:32:02	Reshma Tuladhar:	Thank you
03:32:14	William Telford:	But we can normalize the signals on two different instruments using beads and make their apparent sensitivity similar.
03:32:37	Reshma Tuladhar:	Thanks
03:32:47	William Telford:	This is a good idea if two labs with different instruments are collaborating and need to produce comparable data.
03:34:17	PK Wallace:	Hi Zosia, Interesting talk, thanks, picked up a few things. Question: in setting the voltages we ran each PMT at different voltages as you show and determined the Signal to Noise (SN) ratio back before the Stain Index (SI) had been popularized. I hadn’t considered the negative spread but you are 100% correct in that it will affect the analysis. Do you know how these two indices correlate when doing a voltage titration?
03:36:25	Zosia Maciorowski:	Probably pretty similar, but better taking the SD negative into account. Some of the shortcuts for doing this take into account only the spread of the negative, but I think it’s more understandable if you can actually see the separation.
03:38:26	PK Wallace:	Thanks Zosia. that makes sense and I think what you are suggesting is a better way to look at this. I am going to have to sign off at 9:00 for an ISAC call but will be back tomorrow.
03:40:10	Zosia Maciorowski:	Thanks for getting up at the crack of dawn to be here!
03:46:36	Bikash Dwivedi:	i got a question ... i didn't get what are actually negative and positive population you are talking about.
03:55:57	William Telford:	The negative population is at the low end of the scale (to the left) - the positive population is at the high end of the scale (to the right).  The fluorescence intensity of the marker increases as the scale moves to the right (or up in a 2D dot plot).
03:56:13	Zosia Maciorowski:	The tube contains some cells that don’t have the marker or antigen that the antibody/fluorochrome attaches to, thus are called negative. It also contains cells that do have the antigen or marker which will have the antibody/fluorochrome attached and thus will fluoresce at the wavelengths of the fluorochromes: they are positive. Where they are on the scale is indicative of how many markers/antibody/fluorochrome are on the cell. However all cells have a certain amount of background autofluorescence, so they will show on the scale.
03:56:42	Madan Raj Pandey:	where from we can learn about flowjo software. is this software available in internet?
03:56:44	William Telford:	But it depends on the cells - some antibodies label all the cells (like CD45), some only a small population.  The fluorescence distribution depends on the biology.
03:57:39	William Telford:	You can download 30 day fully functional trial versions of both FlowJo and FCS Express, the two dominant off-line analysis software packages.  Both are very good.
03:57:48	Bikash Dwivedi:	thank you sir for the info.
03:58:08	William Telford:	FlowJo at flowjo.com, FCS Express at denovosoftware.com
03:58:46	Madan Raj Pandey:	Thanks Bill for information.
03:59:22	William Telford:	Tribhuvan University Central Department of Biotechnology should have a FlowJo license  - if it is not working, Prof. Manandhar can contact me nd we will get it reactivated.
03:59:40	William Telford:	We can put them on our FCS Express license too.
04:02:12	prakriti karki:	what's the reason behind using laser light? Can laser light be replaced by other?
04:09:51	William Telford:	We really need lasers for flow!  No other light source puts so much single wavelength light and energy in a small area (the cross section of a cell.  Very old cytometers used arc lamps, but they were much less sensitive than modern instruments.
04:10:20	William Telford:	Fortunately lasers are becoming cheaper.  And even cheap lasers are getting more reliable.
04:11:16	prakriti karki:	Thank you
04:25:51	Lyana Setiawan - Indonesia:	Hello.. thanks for the opportunity to join the workshop 
04:26:03	William Telford:	Please join us tomorrow W 5-5-21 17:00 Nepal time - same link.  Today's video will be posted by tomorrow morning.
04:26:40	Pragati Pradhan:	Thanks Bill for the information.
04:26:58	Bikash Dwivedi:	okay sir thank you
04:27:05	Dibyak Kapali:	thank u for today
04:27:09	Dibyak Kapali:	see ya