This was a great, informative step-by-step video. Thank you for uploading. Quick question - is the interior of the culture flask coated with something to keep the cells adhered? It seems they don't come off until treatment with trypsin?
Hi Donne! Thanks for the comment. The flasks are not coated with the carrier. online-shop.eppendorf.ru/RU-ru/Kulturalnyj-plastik-110320/Rashodnye-materialy-dlja-raboty-s-kulturami-kletok-110321/Eppendorf-Cell-Culture-Flasks-PF-68138.html
As a second question, if you are trying to dissociate spheroids (of mouse astrocytes) embedded in collagen (2.4mg/ml) matrix, what working soln concentration would you recommend for collagenase? Also, would it be beneficial to use a cocktail of collagenase, trypsin, and acutase in this case?
Unfortunately, we have no experience with astrocytes, but your cocktail of enzymes seems to me a tough option for the dissociation of spheroids. I think a mixture of collagenase and acutase will be enough. Good luck, MCC
Hey, thank you for the detailed video. I have one question. When you add collagenase you mention it as 10mL of 60U/mL concentration But, when you are adding it to the small tissue pieces (present in the 50mL tube) you add two things using the pipette. Also, I understand you have prepared 60U/mL collagenase from the stock. Do you add it at that concentration directly to the cells or do you again dilute it while adding? Thank you!
Dear Abhinaba Banerjee, We use ready-made collagenase solution in the laboratory (we use DMEM/F12 basal medium to dissolve the dry enzyme). The second reagent is calcium chloride, which is important for the work of collagenase. Good luck, MCC
@@MedicinalChemistryCenter Thank you for your reply! Also, from the stock to the working concentration do we dilute it in cell culture media? For the stock we use HBSS.
I noticed your bare wrists/arms and moving hands over the open samples - is there no risk of contamination in this kind of work? I'm a total beginner trying to understand the rules :)
Thank you for your question. Collagenase is used to de-internationalize tissue and isolate tumor cells. Calcium ions are essential for collagenase to function.
Woww tq for this step by step video.. Very good effort 👍👏👏
Thank you for hand on practice video.
Thank you
This was a great, informative step-by-step video. Thank you for uploading. Quick question - is the interior of the culture flask coated with something to keep the cells adhered? It seems they don't come off until treatment with trypsin?
Hi Donne!
Thanks for the comment.
The flasks are not coated with the carrier.
online-shop.eppendorf.ru/RU-ru/Kulturalnyj-plastik-110320/Rashodnye-materialy-dlja-raboty-s-kulturami-kletok-110321/Eppendorf-Cell-Culture-Flasks-PF-68138.html
@@MedicinalChemistryCenter thank you for clarifying!
As a second question, if you are trying to dissociate spheroids (of mouse astrocytes) embedded in collagen (2.4mg/ml) matrix, what working soln concentration would you recommend for collagenase? Also, would it be beneficial to use a cocktail of collagenase, trypsin, and acutase in this case?
Unfortunately, we have no experience with astrocytes, but your cocktail of enzymes seems to me a tough option for the dissociation of spheroids. I think a mixture of collagenase and acutase will be enough.
Good luck,
MCC
Hey, thank you for the detailed video. I have one question. When you add collagenase you mention it as 10mL of 60U/mL concentration But, when you are adding it to the small tissue pieces (present in the 50mL tube) you add two things using the pipette. Also, I understand you have prepared 60U/mL collagenase from the stock. Do you add it at that concentration directly to the cells or do you again dilute it while adding? Thank you!
Dear Abhinaba Banerjee,
We use ready-made collagenase solution in the laboratory (we use DMEM/F12 basal medium to dissolve the dry enzyme). The second reagent is calcium chloride, which is important for the work of collagenase.
Good luck,
MCC
@@MedicinalChemistryCenter Thank you for your reply! Also, from the stock to the working concentration do we dilute it in cell culture media? For the stock we use HBSS.
I noticed your bare wrists/arms and moving hands over the open samples - is there no risk of contamination in this kind of work?
I'm a total beginner trying to understand the rules :)
There is no risk of contamination. Most importantly, wash your hands well up to the elbows before starting work.
what is the function of cacl2 and collagenase
Thank you for your question. Collagenase is used to de-internationalize tissue and isolate tumor cells. Calcium ions are essential for collagenase to function.