Cell cell dyanimcs, EdU Index & pH3 Index (Fig. 3A, B & Fig S4A, C)

Juveniles were dissociated in pools of 15 polyps. Prior dissociation were labeled for 30 min with EdU (100uM, 2%DMSO) over several time points after feeding, and then dissociated 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, mOr2 was detected with anti-dsRed (Mouse, Takara Bio Clontech 632543) and goat-anti-mouse Alexa568, pH3 was detected with anti-pH3 (Ser10) (Rabbit, Cell Signaling Technology, 9701) and and goat-anti-rabbit Alexa488.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15个水螅体聚合体中的幼体进行解离。解离前，于喂食后的多个时间点，使用含2%二甲基亚砜（DMSO）的100μM 5-乙炔基-2'-脱氧尿苷（EdU）标记细胞30分钟，随后采用胰蛋白酶（Trypsin）与甲醛进行解离及固定。以1μg/mL的FxCycle Violet DNA染料（Invitrogen）对细胞进行染色；通过Click-iT反应，利用Alexa647对EdU进行标记；使用抗dsRed小鼠单克隆抗体（Takara Bio Clontech，货号632543）及山羊抗小鼠Alexa568二抗检测mOr2蛋白；使用抗磷酸化组蛋白H3（Ser10）兔源多克隆抗体（Cell Signaling Technology，货号9701）及山羊抗兔Alexa488二抗检测pH3蛋白。采用BD LSRFortessa流式细胞仪（BD Life Sciences）完成流式细胞检测；基于DNA染色确定细胞周期各时相细胞，以阴性对照为参照，借助FlowJoV10.8软件（BD Life Sciences）分析Alexa568与Alexa647的荧光信号，以此判定样本中mOr2及EdU阳性细胞。.wsp文件包含FlowJo软件中用于分析的样本信息、门控策略及细胞群数据。.fcs文件为单个流式细胞仪输出文件，均在对应.wsp文件中完成分析。