Mr. Cross, thank you so much for this awsome video. It is really a review and informative for m.. Great presentation and easy to undersand. Kudos from Morocco.
What happens with the phenol red ( from the composition of media): Is the phenol red able to change its color from basic to acidic and then after pH changes from basic to acidic ?
@@funny11744 Phenol red actually can change its color from basic to acidic and then back again! But under two conditions it will not be a suitable component: 1. Multiple cycles of going from acidic to basic can degrade the phenol red over time, making it less effective as an indicator. 2. If the media becomes very acidic (much lower pH), the phenol red might not change back completely even if the pH is adjusted upwards. This is because the cells themselves might be stressed or dying, causing a permanent shift in the media's pH.
While it's possible to count cells without Trypan Blue, this method doesn't assess cell viability. Trypan Blue helps differentiate between live and dead cells, ensuring you're working with a healthy population.
Is it "safe" to pour cells out of the flask? Especially when working with primary cell lines, would it be better to pipette and avoid the flask's lip? or does it not matter?
As you pointed out pouring cells out of the flask is generally not recommended. Pipetting is the preferred method for handling cells, especially primary cell lines which are more delicate. Pipetting allows for precision and reduced contamination risk.
Hi Neil, I am facing contamination continuously in my cultured cell. I checked my media and My insect cell media which i prepared manually using 2x sfc media, gentamycin, P/S ,serum and cell culture graded water. Could you please tell me how can I know that which of any cell culture reagent having contamination or not?? I sprayed alcohol vigorously on the reagent bottles, holders, pipettor, washed my hand, wearing gloves, rubbing my gloves with alcohol, keeping my empty plates and plastic container under the uv inside the hood, still contaminated media... :(
Because he is using anchorage-dependent cells, cells that only grow if they adhere to a surface. The cells are simply doing what they would do in vivo, inside the body. Cells that belong to tissues produce molecules that mediate both adhesion to the surface and cohesion with each other in order to actually form the tissue. On the other hand, red blood cells for example are usually in circulation in our blood so they wouldn't naturally adhere to a surface.
Washing with PBS removes unwanted components from cell cultures (dead cells, debris) and prepares them for further steps (staining, assays) without harming the cells. It provides a clean, consistent environment with the right salt balance for healthy cells.
Yes, breathing over the cell culture can be a source of contamination. You can introduce bacteria or fungi from your mouth and nose. In a sterile lab, wearing a head cover and surgical mask is essential to minimize this risk. Additionally, working in a biosafety cabinet provides further protection by filtering the air and creating a sterile work zone.
@@louisjacobmoon the basic idea of lab meat is to avoid killing animals, so if you are killing an animal to get Trypsin or something out of them so that the cells can feed-on and multiply then it defeats the purpose.
Many now use recombinant Trypsin eg TrypLE www.thermofisher.com/uk/en/home/life-science/cell-culture/mammalian-cell-culture/reagents/trypsin/tryple-express.html?s_kwcid=AL.3652.3..e..o..tryple&ef_id=869904f24fa9112537fe6585ae811767:G:s&s_kwcid=AL!3652!10!76691033548341!76691086026937&cid=bid_clb_cce_r01_co_cp0000_pjt0000_bid00000_0se_bng_bt_pur_con&msclkid=869904f24fa9112537fe6585ae811767 However the foetal calf serum is most certainly animal-derived... There are some new but very expensive non-animal serum replacements
I guess the falcon tubes can be reused if autoclaved no need to incinerate them, I am saying this with respect to universities protocol no idea why its done in professional labs....
The best practice is to avoid any type of cross-contamination during the study. Cross-contamination can be expanded to include chemical contamination that may occur if autoclave is not properly carried out. This is a common practice in many laboratories.
Really great for someone who already has cell culture experience and just needed a quick crash course!
I can't agree more ! Exactly!
In other words, he is being waay to casual going about this to nitpick on. Haha
But still a great video.
This is the best video I have ever seen. Many congratulations, for having put your best effort into making it happen.
Thank you so much this was really helpful. Going to crush my interview now!
Very informative; step by step explanation. This is the perfect video I craved for. Nice work, Cross
This is extremely useful, thanks Neil!
Excellent video, very clear and informative.
This is awesome, thanks! One note for anyone watching: Make sure you don't lay the pipette tip down with fluid still in it like at 11:05
This is the best video have seen. Thank you so much
I love your videos! thank you!
Excellent efforts, very nicely explained
Thank you for the content.
Thank you soo much!!!! That was brilliant
Very nicely explained sir. Thanks a lot..
Thank you very much. This is very explicit.
This is an amazing work! Thank you so much!!
Question : Regarding the stem cell culture in T 75 flask - using media without CO2 : Can we use a closed cap for T75 flask ?
Great vedio for cell culture indeed😊
It was very helpful!! Thanks!
Very nicely explained sir thank you very much 👍
véry good explanation sir
Thank you this was very helpful!
Very informative 👍🏻👍🏻👍🏻
Absolutely great
this is so useful thank you so much for making this!!!!!!!
Very thorough love this
Thank you for this nice video
Great job 👏
Very good video , thanks
Mr. Cross, thank you so much for this awsome video. It is really a review and informative for m.. Great presentation and easy to undersand. Kudos from Morocco.
What happens with the phenol red ( from the composition of media): Is the phenol red able to change its color from basic to acidic and then after pH changes from basic to acidic ?
@@funny11744 Phenol red actually can change its color from basic to acidic and then back again! But under two conditions it will not be a suitable component:
1. Multiple cycles of going from acidic to basic can degrade the phenol red over time, making it less effective as an indicator.
2. If the media becomes very acidic (much lower pH), the phenol red might not change back completely even if the pH is adjusted upwards. This is because the cells themselves might be stressed or dying, causing a permanent shift in the media's pH.
@@kosheeka thank you for information.
Thanks for this video👍
Hi thanks great video , so we was taught to use aseptic technique by spraying anything going in the the fume hood ,
It is always a good practice to keep following it.
When you filling the 96well plate are you doing reverse pappeting?
Thank u so much 🙏😊🙏🙏
REALLY GREAT .
Very helpful 👍
Thank you!
Thank you
thanks so much
An informative video
Very good presentation
I serial dilution for that plating solution?
No trypandblue?😮
Thank you for this video, however I can't find the tutorial for counting cells :(
extremely helpful
Why he put 1.2 instead of 120 ? Someone explain please
Nice sir it help us lot in COVID situation.
Great video!
wonderful and well presented. hope i could an opportunity to work under your supervision and guidance. i am final year student of biotechnology.
very informative and fast. very good technique! Could the counting be made without trypan ? It seems yes ,
While it's possible to count cells without Trypan Blue, this method doesn't assess cell viability. Trypan Blue helps differentiate between live and dead cells, ensuring you're working with a healthy population.
Is it "safe" to pour cells out of the flask? Especially when working with primary cell lines, would it be better to pipette and avoid the flask's lip? or does it not matter?
As you pointed out pouring cells out of the flask is generally not recommended. Pipetting is the preferred method for handling cells, especially primary cell lines which are more delicate. Pipetting allows for precision and reduced contamination risk.
I had the same concern
Does this really follow aseptic practice? I doubt that。
Hi Neil, I am facing contamination continuously in my cultured cell. I checked my media and My insect cell media which i prepared manually using 2x sfc media, gentamycin, P/S ,serum and cell culture graded water. Could you please tell me how can I know that which of any cell culture reagent having contamination or not?? I sprayed alcohol vigorously on the reagent bottles, holders, pipettor, washed my hand, wearing gloves, rubbing my gloves with alcohol, keeping my empty plates and plastic container under the uv inside the hood, still contaminated media... :(
You can autoclave the media if you think any component of the media is causing it
How are the cells adhering to the flask enough so they're not dumped out with the fetal calf serum?
Because he is using anchorage-dependent cells, cells that only grow if they adhere to a surface. The cells are simply doing what they would do in vivo, inside the body. Cells that belong to tissues produce molecules that mediate both adhesion to the surface and cohesion with each other in order to actually form the tissue. On the other hand, red blood cells for example are usually in circulation in our blood so they wouldn't naturally adhere to a surface.
Can I ask if there are any cells in the dmem media that you throw out or all cells only adhere to the flask?
cells adhered to the flask
Until we add trypsin in the culture the cells stick to the flask, but after the trypsinization step, your cells will be in the medium so be careful.
Wait why would you wash the cell to remove the medium with the PBS? I mean medium is required for cell growth, why remove it?
Washing with PBS removes unwanted components from cell cultures (dead cells, debris) and prepares them for further steps (staining, assays) without harming the cells. It provides a clean, consistent environment with the right salt balance for healthy cells.
Is breathing on it adding potential contaminating?
Yes, breathing over the cell culture can be a source of contamination. You can introduce bacteria or fungi from your mouth and nose. In a sterile lab, wearing a head cover and surgical mask is essential to minimize this risk. Additionally, working in a biosafety cabinet provides further protection by filtering the air and creating a sterile work zone.
Sir explain how insulin cells are produced and insulin injection are prepared
Are any animals killed during the extraction of Trypsin from their pancreas ? Or can it be done without killing the animal ?
Why?
@@louisjacobmoon the basic idea of lab meat is to avoid killing animals, so if you are killing an animal to get Trypsin or something out of them so that the cells can feed-on and multiply then it defeats the purpose.
Many now use recombinant Trypsin eg TrypLE www.thermofisher.com/uk/en/home/life-science/cell-culture/mammalian-cell-culture/reagents/trypsin/tryple-express.html?s_kwcid=AL.3652.3..e..o..tryple&ef_id=869904f24fa9112537fe6585ae811767:G:s&s_kwcid=AL!3652!10!76691033548341!76691086026937&cid=bid_clb_cce_r01_co_cp0000_pjt0000_bid00000_0se_bng_bt_pur_con&msclkid=869904f24fa9112537fe6585ae811767
However the foetal calf serum is most certainly animal-derived... There are some new but very expensive non-animal serum replacements
I guess the falcon tubes can be reused if autoclaved no need to incinerate them, I am saying this with respect to universities protocol no idea why its done in professional labs....
The best practice is to avoid any type of cross-contamination during the study. Cross-contamination can be expanded to include chemical contamination that may occur if autoclave is not properly carried out. This is a common practice in many laboratories.
Finally found a cure for my son Sickle cell I’m so glad you came into my life. I pray for long life so you can save more souls,Thanks #druromi .
wow can you share your work or publications?
@@mrx4814 Yes sure