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pretty stupid Q


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#1 Suhas

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Posted 07 February 2011 - 09:13 AM

I've been doing cell culture since yrs but still haven't gotten around to understanding how split ratios work

say i need to split a 100mm dish that is fully confluent

i use 2ml trypsin to detach cells

then i add about 8ml media to inactivate trypsin

now my cells are already diluted 1:10 right?

supposing i add 1 ml of this resuspended cells into a 100mm dish containing 9ml new media. its 1:10 again. so totally its 1:20 dilution. is this right?

but most online protocols don't tell you how much media to use to inactivate trypsin. doesn't that matter at all? i might use just 2 ml of media (which is a 1:2 dilution) or 20 ml media (which is 1:20 dilution). it keeps me confused all the time

suhas

#2 Rnotk

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Posted 07 February 2011 - 04:00 PM

how much medium that you use to inactivate trypsin is not a point.

if you pass from one 10cm dish to ten 10cm dishes, it is 1:10 split.
just think about the growth area before and after the passage.




I've been doing cell culture since yrs but still haven't gotten around to understanding how split ratios work

say i need to split a 100mm dish that is fully confluent

i use 2ml trypsin to detach cells

then i add about 8ml media to inactivate trypsin

now my cells are already diluted 1:10 right?

supposing i add 1 ml of this resuspended cells into a 100mm dish containing 9ml new media. its 1:10 again. so totally its 1:20 dilution. is this right?

but most online protocols don't tell you how much media to use to inactivate trypsin. doesn't that matter at all? i might use just 2 ml of media (which is a 1:2 dilution) or 20 ml media (which is 1:20 dilution). it keeps me confused all the time

suhas



#3 HomeBrew

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Posted 07 February 2011 - 05:33 PM

A 1:10 dilution of a 1:10 dilution is a 1:100 final dilution, not 1:20...

#4 leelee

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Posted 07 February 2011 - 07:42 PM

Your trypsinised and resuspended cells in 10ml are not 1/10...yet...they are just the cells of one dish in 10ml of media....

if you aliquot 1ml into a new dish then you have used 1/10th of the original cells so the ratio is a 1/10 split

if you aliquot 2ml into a new dish, then you have used 2/10th of the original cells so the ratio is a 1/5 split....

You could resuspend your cells in 20ml or 100ml or whatever you like and it doesn't matter, what matters is what proportion of the original cells go into the new dish.

Make sense?

Then how much you top your cells up with in the new plate is completely irrelevant, as it has no effect on the amount of cells present (except to help them grow of course!).

#5 Suhas

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Posted 08 February 2011 - 03:52 AM

got it!

thanks a lot everyone ;)

#6 Chaser

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Posted 11 February 2011 - 05:22 PM

how much medium that you use to inactivate trypsin is not a point.

if you pass from one 10cm dish to ten 10cm dishes, it is 1:10 split.
just think about the growth area before and after the passage.





I've been doing cell culture since yrs but still haven't gotten around to understanding how split ratios work

say i need to split a 100mm dish that is fully confluent

i use 2ml trypsin to detach cells

then i add about 8ml media to inactivate trypsin

now my cells are already diluted 1:10 right?

supposing i add 1 ml of this resuspended cells into a 100mm dish containing 9ml new media. its 1:10 again. so totally its 1:20 dilution. is this right?

but most online protocols don't tell you how much media to use to inactivate trypsin. doesn't that matter at all? i might use just 2 ml of media (which is a 1:2 dilution) or 20 ml media (which is 1:20 dilution). it keeps me confused all the time

suhas


It doesmatter how much media you use to inactivate the trypsin. If you originally had 10mL in the dish, aspirate and add 2mL trypsin and 3mL of media to inactivate, you therefore have the same amount of cells but in half of the original volume. So for a 1:10 split, instead of taking 1mL into a new dish with 9mL media, you would take 500 microliters into a new dish with 9.5 microliters of media.

#7 bob1

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Posted 11 February 2011 - 09:20 PM

It doesmatter how much media you use to inactivate the trypsin. If you originally had 10mL in the dish, aspirate and add 2mL trypsin and 3mL of media to inactivate, you therefore have the same amount of cells but in half of the original volume. So for a 1:10 split, instead of taking 1mL into a new dish with 9mL media, you would take 500 microliters into a new dish with 9.5 microliters of media.

No it doesn't, ratios are independent of volume, whether you have 1 ml or 5 l a 1:10 split is still taking 1/10th of the volume...




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