侧群干细胞（sp细胞）的分离与分选
准备工作：
DF12 加入0.1%BSA，100ml,取10ml冰浴，40ml放入孵箱；其余的放入4度；
DNase I 100*; PI：50ug/ml; Hoechst33342: 1000ug/ml（all from Sigma）
计数板，冰盒；各种离心管，无菌流式管（BD），400滤网（BD）
染色方案：
① 细胞染色：
细胞消化计数，用DF12重悬，调整到1×106/ml浓度，分成三组2管；
37℃DF12洗涤，制备成单细胞悬液；
两组均加入荧光染料Hoechst33342，使终浓度为6 ug/ml，其中CON组再加入维拉帕米，终浓度50 umol/ml；
37℃避光水浴90 min；
冰上10 min终止染色，计数细胞；离心细胞后，用冰预冷DF12洗涤细胞并重悬浮，将分选组调整浓度到1×107/ml以上，必要时加入1倍的DNase I；
上机前再加入PI使终浓度为2 ug/ml. 并且400滤网过滤细胞
② 流式细胞仪检测：
355 nm UV激发光源，610 nm双色短通反射滤镜，450 nm和675 nm边缘长通滤片分别检测散射光蓝光及红光部分，线性模式采集信号. 测量前向散射和侧向散射二维参数图，检测细胞均一性，以Hoechst Red为X轴，Hoechst Blue为Y轴作二维散点图，将低Hoechst Red及低Hoechst Blue且维拉帕米组缺失的区域设定为SP细胞的“门”（gate），计算百分比. 分选出SP细胞及Non SP细胞.
goodell的sp分选
http://sciencepark.mdanderson.org/fcores/flow/files/SP.pdf：
Hoechst 33342 Cell Staining and Side Population Purification Protocol
(see Goodell, M., et al. (1996) J Exp Med 183, 1797-806)
The ability to discriminate Hoechst SP cells is based on the differential efflux of Hoechst 33342 by a multi-drug-like transporter. This is an active biological process. Therefore,optimal resolution of the profile is obtained with great attention to staining conditions.
The Hoechst concentration, staining time, and staining temperature are all CRITICAL.
Likewise, when the staining process is over, the cells should be maintained at 4°C in
order to prohibit further dye efflux.
1) Ensure that a water bath is precisely at 37°C. The medium needs to be prewarmed
beforehand.
2) To thaw cells from the cryovials, put them directly from dry ice into the 37°C
water bath. Once the cells are thawed, wash them once by transfering them to a
centrifuge tube, adding 9 mls of their respective media, and spinning for 5
minutes at 1200 rpm in order to remove the cryo-preservants.
3) Resuspend at 106 cells per ml in pre-warmed DMEM+5% FBS. Mix well.
4) Add Hoechst to a final concentration of 5 µg/ml (a 200x dilution of the stock).
5) Mix the cells well, and place in the 37°C water bath for 90 minutes exactly. Make
sure the staining tubes are well submerged in the bath water to ensure that the
temperature of the cells is maintained at 37°C. Tubes should be mixed several
times during incubation.
6) After 90 minutes, spin the cells down in the COLD and resuspend in COLD
Phosphate Buffered Saline (PBS).
7) All further proceedings should be carried out at 4°C to prohibit leakage of the
Hoechst dye. Add 2 µg/ml of 7-AAD to the suspended cells (a 100X dilution of
the stock provided) and mix about 5 minutes before FACS analysis. This will
allow for the discrimination of dead versus live cells as 7-AAD permeates only
cells that do not have an intact membrane.
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