Hi everyone
I'm new to cell culture, so any help would be much appreciated.
I need to replate 5X10^5 from 10cm dish into each well of 6 well plate. Any ideas of protocols to guid me?
Thank you in advance
HI,
What we usually do for our experiment, we trypsinize and calculate the cells/mL. If we have x cells in 1mL and we want y cells for my experiment, then we would take y/x mL and spin it down. Now if we want to seed (y/x)/6 cells in each well in a 6 well plate, then we would resuspend y/x cells in 6mL media and distribute ImL to each well. We feel it'll reduce pipetting error.
It’s always a good idea to make a bit extra: to have 25% extra have 2ml of your trypsinised solution and add 35.5ml of fresh medium. You may also want to consider that cells will obviously grow so you can always use lower seeding cell concentrations if you're incubating your plates longer before treatment/collection.
Hope this help!!
Hi,
The seeding density / split ratio always depends on your cell line. When you have very common cells, like HeLa etc. you can make a quick google search for a suitable protocol. Usually the companies provide a guide to their cell on how to split them (e.g. Trypsin, EDTA, Accutase ...).
If you are working with a completely new cell line (e.g. patient derived cells), you will have to test out the perfect split ratio for those cells. E.g. you detach your cells (with trypsin for example), spin them down, resuspend in 1 ml of medium and plate them with different ratios, e.g. ranging from 1:3 - 1:20 (check the literature first, this is highly variable for different cell types). Even if you lose a lot of wells, because the density was too low/too high, you will at least have 1 well with a good split ratio. Use this ratio for further splits.
It is more time consuming, but way more precise: Detach your cells, spin them down, count them and seed different cell numbers in your wells. You can then determine the perfect amount of cells/cm².
Hi,
Firstly, what cells are you working with? Some cells can be trypsinised (using trypsin/EDTA mix) to detach from the culture vessel, some grow in suspension and so do not need to be detached. Others cannot be detached using T/E and so need to be scraped off using a cell scraper.
Please see the website below for general basic tissue culture protocols - it's basically what I was going to type in so saves time typing a long essay! It has nearly all you need to get by with tissue culture work.
Hope it helps!
https://www.phe-culturecollections.org.uk/technical/index.aspx
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