外周血流式

Sophia

请问，有谁做过大鼠外周血的流式检测？

iseeyou1210

这个是我用得protocol,来源于Jax labs.
4 Q( O6 r% Q0 l5 U: _0 K有一点要注意的是,我一般lysis red blood cell with 2 round of Ack buffer!  @2 U/ Y: B0 r& u" Y( r
good luck.
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FACS Analysis Using Peripheral Blood Cells! S* B9 n8 b; b  ?# m$ b; \4 m
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.* w  M" o! q+ O9 z: s
Remove 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.)) N- m% [* u' q1 L8 N
Cells 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).6 k) K8 `( j  O* h9 e3 @  t
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.6 S& @, b' g) {. ]. K
Incubate for 30 minutes on ice., W4 w* a+ F, ^$ Z' E
Wash cells 2-3x with FACS buffer and suspend in 200-300 microliters FACS buffer for analysis.
7 q" P0 H; W% d$ v, p# q4 ?For live/dead discrimination, add 10 microliters propidium iodide (PI) solution (stock=10 micrograms/ml). If fixing cells before analysis, do not add PI.

iseeyou1210

我做过小鼠的,效果还可以.
( F; E/ d" }9 l. Z9 r" N5 C# t7 h/ R4 r我觉得小鼠和大鼠的差不多把.