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.0 u0 i, `9 z3 T
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.
$ z" }1 ^9 ^. ]1 _& `- a) h9 K1 v有一点要注意的是,我一般lysis red blood cell with 2 round of Ack buffer!
- g2 W' f8 P7 v) i* G$ ^* Vgood luck.
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FACS Analysis Using Peripheral Blood Cells
: c( }  b# ]+ RCollect 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., V/ d! ]4 B; q) w/ X0 ^  l
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.); b8 @6 l- z' u" X
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).
& f+ `' Z1 J6 \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.2 H% ?$ q+ ?, `; B, n
Incubate for 30 minutes on ice.- A5 Y, j6 r; P/ b8 w. P
Wash cells 2-3x with FACS buffer and suspend in 200-300 microliters FACS buffer for analysis.
  }& d  f5 b. Q7 V( t8 p! I! ]5 N5 M& bFor live/dead discrimination, add 10 microliters propidium iodide (PI) solution (stock=10 micrograms/ml). If fixing cells before analysis, do not add PI.