Youtor
Harvesting cell culture basics and practical tips
>> YOUR LINK HERE: ___ http://youtube.com/watch?v=E7weEhLno2w
It’s always harvest season in the lab! Because “harvest” is a term we use to describe separating cells we’re growing in cell culture from the media (liquid food) we’re growing them in and keeping the cells to do fun things with. Here’s the basics of how we can harvest cells - both suspension cells (cells growing suspended in liquid, often shaking in an Erlenmeyer flask) - and adherent cells (cells growing while stuck to a plate or dish). • blog: https://bit.ly/harvesting_cells • For suspension cells, we typically centrifuge them to pellet out the cells, pour off the media, optionally wash them by resuspending them in clean liquids often PBS), then freeze the pellet or resuspend it in the buffer we plan to later lyse them in, containing protease inhibitors to protect the stuff that will come out of the cells when they lyse (break open). • note: You need to be much gentler with animal cells than bacterial ones. Bacterial cells have more protection, thanks to their cell walls, but animal cells are delicate, so take care to avoid premature lysis. It’s a good thing bacteria can take more stress because we have to use higher centrifugation speeds to pellet them out since they’re smaller than animal cells. Typically we use ~4000-5000 x g to spin down bacterial cells ~100-1000 x g to spin down mammalian cells (use the low end (100-500) if you want the cells to stay alive, higher end when you’re just trying to harvest them to lyse or something and would rather have the firmer pellet and better chance to make sure you collect all the cells). • more on centrifugation: blog: https://bit.ly/lab_centrifuges ; YouTube: • Working with laboratory centrifuges a... • more on pelleting: https://bit.ly/pelleting_practical ; YouTube: • Pelleting tips and tricks • For adherent cells, we have to get them off the dish - but first we take advantage of the fact that they’re stuck… We can simply aspirate (suck off) the media, rinse them with PBS, suck that off, then, now that they’re clean, scrape them off or use an enzymatic method like trypsinization to sever their connections to the dish and to other cells. • more on trypsinization: blog: https://bit.ly/trypsin_edta ; YouTube: • Cell detachment with trypsin/EDTA: th... • more on cryopreserving cell stocks: blog form: https://bit.ly/cryopreserving_cells YouTube: • Cryopreserving cell stocks • more posts on cell culture: https://bit.ly/cell_culture • more on osmosis and tonicity: blog form: https://bit.ly/osmolarityandmore ; YouTube: • Osmolarity, tonicity, predicting so... • more on cell culture media: blog: https://bit.ly/cell_culture_media ; YouTube: • Mammalian cell culture media basics • • more (hopefully) helpful random practical lab tips tricks: https://bit.ly/lab_tricks_page • • more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0 or search blog: http://thebumblingbiochemist.com
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