00:34:33 William Telford: Previous workshop content: https://www.cytometryunderground.com/TU_Flow_2021.html 00:34:50 krishna: Participants, Please raise your hands if you have any questions or any issues related to the class 00:39:33 William Telford: Paul Edward Hutchinson contact - lsipeh@nus.edu.sg 00:58:35 Ramanuj: Could you elaborate on the vortexing speed, duration, method (e.g pulse) to get the maximum stained cells eventually 00:59:46 William Telford: If you vortex, do it very gently. Cells can be damaged by excessive mixing. Very short pulses (102 seconds), low intensity. 01:00:25 William Telford: If you need to mix strongly to resuspend your cells, there may be other problems with your cell suspension. Ceels should be able to easily resuspend. 01:01:17 Ramanuj: Thank you so much. 01:02:33 Zosia Maciorowski: Mix it gently after decanting as a pellet, it will break up more easily than when you add volume. You can also flick the bottom of the tube gently with your finger to resuspend before adding buffer 01:05:03 Ramanuj: Thank you zosia mam, your slide was very informative. The query is with respect to before the wash step in lysis step/sample addition. Like the number of times it required for vortex for the better result 01:06:52 Zosia Maciorowski: Maybe we can have a discussion with Paul at the end, there are a lot of different opinions on vortexing. 01:07:18 Ramanuj: sure, I would appreciate it 01:20:57 Raju Kumar Dubey: slide is not changing 01:21:57 Pragati Pradhan: The slides are changing in our side. 01:22:47 Riya Shrestha: Query about washing and the graphs related to one wash, two washes: I understand that washing removes the unbound antibodies, meaning that the number of negative sample decreases. And that y-axis in the graph represented cell count. If the number of negative sample decreases after washing, why does the peak height of the negative sample in the graph increase with increasing number of washes? 01:23:04 William Telford: Are the slides changing for you now! 01:23:25 Reshma Tuladhar: Yes it is. 01:26:08 Zosia Maciorowski: It’s more a case of the negative peak being lower, cleaner, tighter and higher, with less spread after washes. The total number under the negative peak should be about the same 01:26:31 Lize Engelbrecht: Reshma, remember the flow cytometer analyses all the cells, not the unbound antibodies. Washing removes the antibodies binding non-specifically, so if there were cells with some antibodies attached non-specifically, the next wash will move that cell from the positives to the negatives. 01:27:08 Zosia Maciorowski: Thanks Lize, nice to see you here! 01:28:19 Lize Engelbrecht: Glad to be here :) 01:28:27 Riya Shrestha: Thank you very much for the clarification! 01:28:40 Reshma Tuladhar: Thanks 01:42:29 krishna: Liz, Welcome to Nepal workshop 01:50:27 Ramanuj: For antigen specific T cells, why there is difference in two different collection tubes: Heparin and EDTA. how is it gone affect the result? 02:00:10 Ramanuj: Thank you very much Hutchinson and paul sir 02:02:55 suman gautam: is heparin only option can we use sodium citrate? 02:03:02 Reshma Tuladhar: Does the temperature affect the binding of labelled antibody to the antigen? 02:03:49 Gorakh Giri: How ICS can be used to study infections from co-stimulated culture of different strains of mycobacteria? Please, comment on selection of kind of cytokines? 02:04:31 krishna: If whole blood assay is still good for stimulation of cells to see expressions, is it worthy to isolate specific cell and go for stimulation to read expressions? 02:10:37 Reshma Tuladhar: Thank you so much for the answer. 02:11:21 suman gautam: During HLB-27 detection by flowcytometry we use EDTA blood . Does it depends on the type of cell we detect or antigen? 02:12:39 Gorakh Giri: Thank you 02:15:56 Lize Engelbrecht: I have to go again, but was good to see you and join the workshop! 02:16:19 Zosia Maciorowski: Thanks Lize! 02:16:47 William Telford: Yes, thanks Lize! 02:18:23 Shova Shrestha: Thank you Paul for the insightful presentation 02:30:13 William Telford: Today's video and referenced papers will be posted at: https://www.cytometryunderground.com/TU_Flow_2_2021.html 02:31:08 William Telford: Tomorrow Thursday 6 May will be a live FACSCalibur data acquisition presentation. 17:00 Nepal time, same Zoom link as today. 02:32:01 Pragati Pradhan: Thanks a lot Bill for the information. 02:35:10 Dibyak Kapali: Good to be here 02:35:18 Dibyak Kapali: Thank you for today! 02:35:23 Bikash Dwivedi: thank you 02:35:42 Pragati Pradhan: Thanks a lot Paul for your elaborative presentation. 02:35:51 Madan Raj Pandey: Thanks everone. 02:35:59 Evance Pakuwal: Thank you! 02:36:11 Amulya Baral: Thank you!