外周血流式

Sophia

请问，有谁做过大鼠外周血的流式检测？

iseeyou1210

我做过小鼠的,效果还可以.
& h: t& |4 [. D+ O% J( w+ O  p( b我觉得小鼠和大鼠的差不多把.

iseeyou1210

这个是我用得protocol,来源于Jax labs.! i6 r$ n2 [, j5 u7 p& B3 r
有一点要注意的是,我一般lysis red blood cell with 2 round of Ack buffer!& A6 P  N! {7 g- @6 x
good luck.
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FACS Analysis Using Peripheral Blood Cells% ~/ h& P8 I1 ~- f9 d) Y! Z
Collect blood (75 microliters) into 1ml PBS containing 5 microM EDTA (10 microliters of 0.5 M stock) and mix immediately to prevent clotting. Keep tubes on ice.
2 x( }; k* I7 SRemove RBCs from samples. This can be accomplished by several means. At The Jackson Laboratory, RBCs are lysed using either Gey's solution or a buffered ammonium chloride (ACK) solution. (Alternatively, Becton-Dickinson sells a product called "FACS lysis buffer" that is used after the staining protocol to lyse RBCs and fix the cells.)
4 v& a2 S' K! U0 Z/ ACells should be washed 2-3x with FACS buffer (PBS supplemented with either 1% BSA or 5% FBS and containing 0.05% NaN3). Suspend the pellet from the final wash in 50 microliters FACS buffer (or more if more than one analysis is to be done on a single sample - up to three separate staining reactions can be set up from a single sample).. W/ |. w* N  s; U4 D/ c/ O# \  A; J
Add 50 microliters of cell suspension to 10 microliters of antibody solution and mix gently. You will need to determine the proper concentration for each antibody used.
/ g6 ~6 s; J) ~' Z# cIncubate for 30 minutes on ice.
: v3 Q( L, K/ i  [. }Wash cells 2-3x with FACS buffer and suspend in 200-300 microliters FACS buffer for analysis.4 t5 o: i6 }& P( d
For live/dead discrimination, add 10 microliters propidium iodide (PI) solution (stock=10 micrograms/ml). If fixing cells before analysis, do not add PI.