Thawing, Passaging and Freezing Cells (Biology 513 - Animal Tissue Culture)

General Information for everyone else:
This video has at least 3-5 mistakes, so I wouldn't even bother fallowing techniques in this video unless someone already knows them and just wants for fun to spot them. Most of the mistakes are found in passaging technique. (Best way is to use lab manual for more accurate steps)
Now to the excuses:
This video was made between the classes of the week of the midterms, plus we had time constrain on top of the due date right at our door steps. So, I would watch this video with caution. Maybe, later I will add annotations tell you guys were the mistakes were made.
Moreover, we do however know the technique steps well and what degree C is what, but for some reason during the video we were saying gibberish (apparently doing steps is not the same as doing them and voicing them) The few steps were excluded and that was due to mistakes in post production.
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The techniques commonly practiced in cell culturing are thawing (resuscitation), passaging (splitting, subculturing) and freezing cells for later use.
The techniques are demonstrated by the students of Biology 513 - Animal Tissue Culture class at California State University of San Bernardino (CSUSB)
#CSUSB
#tissueculture
#stemcells
#mESC
#lab

[@elizabethcromwell2987]

This is a great learning /teaching video, with the text boxes added to correct mistakes. thanks!

[@spectator59]

"you need safety" ... proceeds to handle LN2-frozen vials with his bare hand. 👍

[@EmeritusFX]

Great video. Never knew that opening the biosafety cabinet door wide open cannot trigger the alarm

[@muhammadjahanzaib1832]

Beside the washing the video was really great. I have one suggestion you should also make a video on judging the confluency of cell culture.

[@saadhasib9653]

Great stuff! Appreciate your annotations too, thanks guys. Only thing I'd add is that it'd have been nice to see what you saw under the microscope as well. Maybe in your future videos eh?

[@saadhasib9653]

Or you could link to the pictures in the video as well actually even now

[@omorugged1]

Alex Seb and co, you tried but with some errors. The guy that demonstrated passaging had many errors, so also others. Avoid bubbles and work slowly yet steady

[@JYOtiRaNJanMANgaRaj]

🙏 Thank you so much ❤❤❤❤

[@vinaykumar-xp3uz]

Alexy harvested cells without trying EDTA?

[@linchen1910]

Guys,Thank you very much. you let me know some helpful knowledge s. Great job！

[@الدكتورهالجامعية]

Thank you

[@science_mbg]

Using t25 flask instead of 15ml Falcon is interesting really

[@keshikesh8667]

Really helpful video. Thanks for uploading

[@abbuambalantota]

Good Job.. Thanks for the Video and Educating

[@nitheshm]

while freezing the cells u haven't trypsinized the cells? and what happens if we dont know the cells in the T-flask and then we need to freeze the cells with certain amount of cells?

[@ihaveLight]

Nithesh Moria Hi, thank you for your question! In this case June counted and passaged cells into two flasks, so I just ended up freezing cells from one of his flasks, so at that moment cells were free floating and there were just the right amount in the flask that we needed to freeze (June counted cells in the second part of the video). In this case we didn't need to trypsinize the cells as they weren't attached, but you are right we do need to typsinize and count the cells before freezing!

[@JoeyHumble]

-37C water bath? Surely water will be frozen at -37C?

[@ihaveLight]

Yes, that is correct it should had been +37C! General Information for everyone else: (probably should add this too into the video's description) This video has at least 3-5 mistakes, so I wouldn't even bother fallowing techniques in this video unless someone already knows them and just wants for fun to spot them. Most of the mistakes are found in passaging technique. (Best way is to use lab manual for more accurate steps) Now to the excuses: This video was made between the classes of the week of the midterms, plus we had time constrain on top of the due date right at our door steps. So, I would watch this video with caution. Maybe, later I will add annotations tell you guys were the mistakes were made. Moreover, we do however know the technique steps well and what degree C is what, but for some reason during the video we were saying gibberish (apparently doing steps is not the same as doing them and voicing them) The few steps were excluded and that was due to mistakes in post production.

[@deepthi8586]

***** Dont you need to wash the flask with PBS after discarding the medium (serum) and before you adding the trypsin? presence of cell medium will actually makes cells not to detach?

[@ihaveLight]

Deepthi Gurajada Good catch. That is correct, the cells should had been washed by PBS before addition of trypsin, any presence of medium (serum) in the flask will deactivate trypsin. I will re-watch the video and correct any mistakes I find.

[@deepthi8586]

***** It would have also been good, if you explained why you incubated cells with Trypsin only for 15-20 seconds and not 4 -5 minutes. Well, anybody who works with cell culture would know that it all depends on what type of cells culture you are working with.

[@ihaveLight]

Deepthi Gurajada I will do that, thank you for suggestion, if you have any other suggestions please feel free to let me know!

[@sepidehesmaeily]

I would like to like 👍you, but the big trouble is that you never maintain sterile conditions for this sensitive technique

[@vivekkumarsingh308]

Sir, can you give me protocol of cell culture.... (Plant & Animal )

[@HassanSalehabadi]

helpful video tnx

[@LITG7000]

ppl still use trypsin?

[@bmxderek10]

just wondering... Why is the sash so high?

[@ihaveLight]

+bmxderek10 Sash?

[@bmxderek10]

the windowed divider between you and everything inside. For cell culture I've only ever seen them with an 8 inch opening.

[@ihaveLight]

+bmxderek10 That is correct it shouldn't be open more than 8 inches. The reason we had it open so high is due to camera recording its own reflection from the window/glass instead of what was happening inside the hood. Nice catch :)

[@geneleu4919]

why I can't watch the video？

[@MelomaniacEarth]

so much of pipette needed🙄🥱

[@7killer778]

what was this. explain rationale

[@ritugupta9834]

That's a fantastic video 👏🏻👏🏻👏🏻👏🏻👏🏻👏🏻

[@OmegaPsiPhi0]

The guy at the end is like my perfect bf 😆

[@sahar9314]

You are very harsh and not showing how you resuspend.

[@dinaaly8401]

great

[@하늘-m2j2b]

시청완료!

[@pijushkantikhanra5260]

Didn't add PBS before adding trypsin.......hahahahaha...

[@ihaveLight]

I would not go off this video as we only had few hours to make, edit and submit the video! This video has many errors!

[@ihaveLight]

Also, I would recommend watching this video on your laptop, in desktop mode you can see our commends in the video outlining what we did wrong. You can’t see our commentaries in the video if you are watching it on phone/tablet.

[@jingyiqin3974]

艾玛，他们都挺萌的哈哈哈哈哈哈哈:)

[@nakorisilani2352]

You should change the title to "Everything you should never do in a tissue culture lab." 🙄

[@xxxfreshman]

Why do you use expensive flasks? And why do you not put the trypsin as pbs 0.05% trypsin on it and after you put it on you remove it directly, and hold the flask/dish on a warm plate for 3-4 minutes, and than resuspent it with new media? And than count ........

[@science_mbg]

we are doing it like that :D