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Counting number of cells - (Dec/25/2017 )



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? 



-Mad Researcher-

1) Confluency is something you learn with experience. The best way is to get someone to show you what they consider 100%, 70%, 50% confluent for the cells they are working on. There is a bit of variation with different cell types, and some types such as neurons are very tricky to work with, but practice is the only way to go.


2) There are a ton of posts on here about this very topic: have a look at the attachment in my reply to this topic:


3) Just like making dilutions for a solution - C1V1=C2V2. Just make sure that you are resuspending your cells fully before making the dilution, and while plating - they settle out fairly quickly.


4) This depends on the transfection reagent you are using. Check the manual for the reagent you have for specifics. Generally the process involves making a dilution of DNA in medium (often serum free), then adding transfection reagent (volume based on amount of DNA), mixing, incubating for a few minutes, then adding to cells. Some protocols/reagents call for making a medium/transfection reagent mix, then adding DNA.