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Thread: Help with Cell Culturing

  1. #1 Help with Cell Culturing 
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    Nov 2013
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    Greetings everybody.

    This topic addresses the discussion of recommended surface area and volumes for cell culture vessels.

    I am relatively new in the field of cell culturing and would really be thankful if anyone is willing to share their cell culturing tips/techniques in this thread. I am constantly being bugged by the question of the volumes of cell culture reagents when I am doing cell passaging, seeding or even freezing. Maybe I can start with cell passaging, followed by seeding and then freezing.

    I am currently culturing H9c2 cells and just completed one passage. Is it safe to say that there is no hard and fast rule when adding say, PBS, trypsin and culture medium?

    I started by thawing 3 ml of cells and adding 7 ml of culture medium on Day 1 (Fri), allowing it to grow over the weekend. Day 4 (Mon) is when I will be doing cell passaging. So basically, I would aspirate the 10 ml of cell suspension, wash with PBS (6 - 8 ml?) followed by the addition of (3 - 4 ml?) trypsin before neutralisation with (3 - 7 ml?) of culture medium before centrifuging in a Falcon tube and transferring to a new culture plate. Before centrifuging, I would remove half the volume of cell suspension as I am doing a 1 : 2 passage. The reason for the question marks is that I am uncertain about the preferred/recommended volumes of these reagents.

    Does anyone have an idea of these volumes and also, if possible, please do share your cell culturing tips and how you get desired results for your cell culture work. It would be really helpful if you could also post microscopic images of your cell culture work with labels and magnification.

    Thank you!


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