Counting number of cells

Go to the profile of Chinmaya Sadangi
Dec 25, 2017
0
3

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

3 Comments

Go to the profile of relaxin
relaxin 5 months ago

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

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

Go to the profile of Chinmaya Sadangi
Chinmaya Sadangi 5 months ago

Thank you.

Can you please help me with question number 1 and 3?

Go to the profile of relaxin
relaxin 5 months ago

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.