I am using a magnetic separation based isolation kit for mouse neutrophils and the protocol mentions 2 incubation on ice for 15 min each. I am aware that low temperature pre-activate the neutrophils. So my questions -

1. What does pre-activation entail? What downstream assays are affected?

2. How long should they be allowed to recover at 37 deg C, after isolation at low temperature?

3. What is the maximum length of time neutrophils can be sustained in culture on Poly L Lysine coated plate?

4. I am planning to use RPMI 1640 supplemented with 10% HI-FBS and 1% PS. What media worked for you?