How to prepare different density gradients using percoll for sperm separation?
Answers
If you use 100% Percoll.. Firstly you have to prepare 10x Sperm TALP solution (you can this solution from an other sperm salt diluter or PBS the way is simple you add pure water 1/10), then you can diluted with 1:9 10X SP TALP and Percoll, respectively. It is 90% Percoll. If you want to 80%, there is a simple way ... You have to dilute 90% Percoll with normal SP-TALP with "cross access". In this way I calculated for you. you need 8 xx(ml) 90% Percoll and 1 xx(ml) SP TALP or other diluter. You have %80 Percoll. (An other words, you need 8 fold 90%percoll what your diluter volume is... For example you use 0,5 ml SP talp then you have to add 4 ml 90% Percoll)... And You can prepare other concentrations of Percoll in this way or you can diluted 80% Percoll with SP talp. If you want 60% then you have to diluted 6 ml 90% Percoll with 3 ml SP- TALP... If you want 40% Percoll you have to dilute 4 ml 90% Percoll with 5 ml SP TALP. At the end if you want 20% Percoll you have to dilute 2 ml 90% with 7 ml SP TALP... If you want to know why you can not use directly Percoll, the answer is percoll's osmotic presure equal nearly zero mmol/L...