How many cells do I have, and how much do I put in each well?
Count cells and seed a plate
Counting on a haemocytometer, and turning that count into a seeding plan.
Counting is a two-part job and the second part is where the surprises are. The arithmetic is a division: cells counted, divided by the squares you counted over, divided by the volume above one square, times whatever dilution you made. For a Neubauer improved chamber that last figure is 10⁴, which is where the familiar multiplier comes from — but a Fuchs–Rosenthal chamber is twice as deep, so using the same number doubles your answer.
The part usually left out is precision. Cells settling into a chamber are a Poisson process, so counting n of them carries an uncertainty of about √n whatever you do. Count 100 cells — the number most protocols ask for — and the 95% interval is roughly ±20%. Getting to ±10% takes around 400. That does not make counting useless; it makes a 15% difference between two flasks not worth reacting to.
Seeding then works backwards from a density, usually cells per cm². Multiply by the growth area of the vessel to get cells per well, and prepare one bulk suspension at the final density rather than dosing each well separately. Dispensing an equal volume everywhere is what makes a plate even.
Trypan blue is normally mixed one to one with the sample, which is a two-fold dilution. Forgetting it halves every number downstream, and the result still looks entirely plausible.