Counting number of cells

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Counting number of cells

Postby newbie17425 » Dec 25 2017 1:08 pm

Hi,

I am new to cell culture and require some help regarding understanding confluency, cell counting, and transfection.

1) I would like to know how can we determine the % of cell confluency before or after sub-culturing?

2) How do I count the number of cells?

3) How do I "Plate 5 x 104 cells per well in 0.5 ml of complete growth medium"?

4) If you have a protocol for cell transfection, could you please share it? How do I measure transfection efficiency? Do I always have to do an immuno to determine the efficiency?

Thanks
newbie17425
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Re: Counting number of cells

Postby relaxin » Dec 26 2017 11:38 am

Nowadays you can do Google search for YouTube videos on any topic. Here is one for counting cells:
http://www.abcam.com/protocols/counting ... ocytometer

For transfection, you can just buy commercial transfection reagent and follow their protocol.
Retired academic researcher. Mention of a specific product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
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Re: Counting number of cells

Postby newbie17425 » Dec 27 2017 12:53 pm

relaxin wrote:Nowadays you can do Google search for YouTube videos on any topic. Here is one for counting cells:
http://www.abcam.com/protocols/counting ... ocytometer

For transfection, you can just buy commercial transfection reagent and follow their protocol.


Thank you.

Can you please help me with question number 1 and 3?
newbie17425
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Re: Counting number of cells

Postby relaxin » Dec 29 2017 12:57 pm

1. I normally determine the % cell confluency by "eyeballing", which is highly subjective. However, this is not critical. When you see there is no empty spaces between cells, it is 100% confluent. It is time to split the culture. It often takes less than 24 hours to go from 50% to 100% confluency.

3.Once you trypsinize the cells, make dilutions and count the cells, you will know how many cells per ml in your original cell suspension. You just adjust the cell concentration in the original cell suspension to 10^5 cells per ml (i.e. double the concentration required) with complete growth medium, and add to a new multiple well plate at a rate of 0.5 ml per well. To make sure all wells have the same number of cells (they tend to settle while your are pipetting), invert the tube a few times before you pipet up the cell suspension.
Retired academic researcher. Mention of a specific product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
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