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?
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: http://www.protocol-online.org/forums/topic/5838-cell-counting/#entry55266
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.