cEdU Index (Fig. 5G, H) - Rapamycin treatment during Starvation

Juveniles were dissociated in pools of 15 polyps. Polyps were continuously labeled with EdU (100uM, 2%DMSO) before dissociation, and fixed using Trypsin/formaldehyde. Cells were stained with 1μg/ml FxCycle violet DNA dye (Invitrogen), EdU was labelled with Alexa647 using a Click-it reaction, and mOr2 was detected with anti-dsRed (Rabbit, Takara Bio Clontech 632496) and goat-anti-rabbit Alexa568.Flow cytometry was performed a BD LSRFortessa (BD Life Sciences) and the mOr2 and EdU positive cells in samples were determined based on Alexa568 and Alexa647 fluorescence in comparison with the negative controls on cells within the cell cycle (based on DNA staining) using FlowJoV10.8 (BD Life Sciences)..wsp files contain the samples, gating strategy and cell populations used for the analysis in FlowJo.fcs files represent the individual flow cytometry output files that were analysed in the .wsp file

将幼体于15个水螅体的集合体系中解离。解离前，持续使用EdU（100μM，2%二甲基亚砜）标记水螅体，随后以胰蛋白酶-甲醛混合液固定细胞。采用浓度为1μg/ml的FxCycle Violet DNA染料（Invitrogen）对细胞进行染色；通过Click-it反应，以Alexa647标记EdU；使用抗dsRed兔源抗体（Takara Bio Clontech，货号632496）及山羊抗兔Alexa568二抗检测mOr2蛋白。采用BD LSRFortessa流式细胞仪（BD生命科学）开展流式细胞检测；以DNA染色结果确定细胞所处的细胞周期阶段，以阴性对照为参照，借助FlowJoV10.8软件（BD生命科学）分析Alexa568与Alexa647的荧光信号，以此确定样本中mOr2和EdU双阳性细胞的占比。.wsp文件包含FlowJo软件分析所用的样本信息、门控策略及细胞群分类方案；.fcs文件则为该.wsp文件中所分析的单个流式细胞仪输出原始文件。