準備工作：
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）
計數板，冰盒；各種離心管，無菌流式管（BD），400濾網（BD）
染色方案：
① 細胞染色：
細胞消化計數，用DF12重懸，調整到1×106/ml濃度，分成三組2管；
37℃DF12洗滌，制備成單細胞懸液；
兩組均加入熒光染料Hoechst33342，使終濃度為6 ug/ml，其中CON組再加入維拉帕米，終濃度50 umol/ml；
37℃避光水浴90 min；
冰上10 min終止染色，計數細胞；離心細胞后，用冰預冷DF12洗滌細胞并重懸浮，將分選組調整濃度到1×107/ml以上，必要時加入1倍的DNase I；
上機前再加入PI使終濃度為2 ug/ml. 并且400濾網過濾細胞
② 流式細胞儀檢測：
355 nm UV激發光源，610 nm雙色短通反射濾鏡，450 nm和675 nm邊緣長通濾片分別檢測散射光藍光及紅光部分，線性模式采集信號. 測量前向散射和側向散射二維參數圖，檢測細胞均一性，以Hoechst Red為X軸，Hoechst Blue為Y軸作二維散點圖，將低Hoechst Red及低Hoechst Blue且維拉帕米組缺失的區域設定為SP細胞的“門”（gate），計算百分比. 分選出SP細胞及Non SP細胞.
goodell的sp分選
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.