Preparation of Mouse Neutrophils

实验概要

Preparation of Mouse Neutrophils

实验步骤

Mice：8-16 weeks old male

Prewarm buffer to room temperature

Buffer A : Ca²  /Mg² ‐free Hank’s buffered saline solution [HBSS; Invitrogen, Grand Island, NY] containing 0.1% bovine serum albumin and 5 mM HEPES.

Percoll stock solution: the stock Percoll solution  is made by adding 1 ml of 1.08 M NaCl to per 7 ml Percoll; store at 4  degree for less then 3 months.

81, 62, 55, 50, and 45% Percoll solutions: the stock solution is mixed with HBSS (Ca²  /Mg² ‐free ) vol by vol to make 81, 62, 55, 50, and 45% Percoll solutions.

1.       Bone marrow cells are flushed from the femurs using buffer A,

2.        Red blood cells are lysed by incubating the bone marrow cells with a  RBC lysis buffer, and stop by adding 10ml buffer A,

3.       The cells are spun down, washed twice with buffer A,

4.       Resuspended in 3 ml of 45% Percoll solution,

5.        A Percoll density gradient is prepared by layering successively 2 ml  each of 62, 55, and 50% Percoll solutions made in Ca²  /Mg² ‐free HBSS on the top of an 81% Percoll solution (3 ml) as depicted in a 15‐ml polystyrene tube,

6.       Centrifuged at 1600g for 30 min at room temperature in a swinging bucket rotor,

7.        The mature neutrophils that are banded between the 81% and 62% layers  can be harvested by inserting a Pasteur pipette on the top of the cell  layer and gently sucking the cells into the pipette,

8.       The cells are immediately diluted into 10 ml of fresh buffer A, washed twice, and resuspended in HBSS containing Ca²  /Mg² ,

9.       The number of cells is determined by counting with a hemacytometer.

Note:

1.       It is important not to apply the brake, because it will disrupt the gradient.

2.       We generally obtain 3–5 million cells per CD‐1 mouse