Rhabditis sp. SB347 males attracted to unablated animals (wild type), and ablated animals for precursors of germline (Z2 & Z3), vulva precursor cells (VPC) and precursors of somatic gonad (Z1 & Z4)

Data from figure 3e. To quantify the degree of attraction of worms to conditioned medium produced by different sexes, we used chemotaxis assays. Conditioned medium (henceforth referred to as ‘supernatant’) was generated by placing 5-10 worms in 100 µL of M9 buffer for 12 h at room temperature. The supernatant was stored in 10 µL aliquots at -20 °C until required. The assay was performed either on a microscope slide with a layer of 1.5% agar placed on top of an empty plate, or on a 6 cm NGM plate. 3 µL of supernatant was added on one side of the slide and 3 cm apart from the control drop that just contained M9. 15 young-adults were placed on the midline between the spots. After 30 min, worms were scored on the basis of their location. The values for the chemotaxis index (CI) were calculated with at least ten replicates for each condition. To calculate the CI, the number of worms attracted to the test spot were subtracted by the number of worms in the control spot, and divided by the total number of males assayed. A CI of one indicates that all worms are attracted to the chemical in the test spot, whereas a CI close to zero indicates no attraction. Gonadal (Z1 – Z4) and vulva precursor cells (P5.p, P6.p and P7.p) of mid- L1 animals (females or hermaphrodites) were ablated using laser microsurgery. For ablation, worms were placed on agar pads on microscopic slides. Worms were recovered after ablation using a drop of M9 buffer and transferred to a normal seeded plate. Two days after recovery, laser ablated adult worms were used to collect supernatant for attraction assays本数据来自图3e。为定量评估线虫对不同性别个体所产条件培养基的趋化程度，我们采用了趋化性实验（chemotaxis assays）。条件培养基（conditioned medium，后续简称上清液（supernatant））的制备方法为：将5~10条线虫置于100 μL M9缓冲液中，于室温孵育12小时。将上清液以10 μL为单位分装，于-20℃保存备用。本实验可在两种载体上开展：一是在空平板上铺有1.5%琼脂层的载玻片，二是直径6 cm的NGM平板（NGM plate）。在载玻片的一侧滴加3 μL上清液，与仅含M9缓冲液的对照液滴相距3 cm。将15条青年成虫（young-adults）置于两个液滴之间的中线上。30分钟后，根据线虫所在位置进行计数。每个实验条件均设置至少10次生物学重复，以此计算趋化指数（chemotaxis index, CI）。趋化指数的计算公式为：（被试验液滴吸引的线虫数 - 对照液滴中的线虫数）除以受试雄性线虫的总数量。当趋化指数为1时，代表所有线虫均被试验液滴中的化学物质吸引；而当趋化指数接近0时，则代表无趋化吸引现象。使用激光显微手术（laser microsurgery）消融中期L1阶段的线虫个体（雌性或雌雄同体）的生殖腺前体细胞（Z1~Z4）与外阴前体细胞（P5.p、P6.p和P7.p）。进行消融操作时，将线虫置于载玻片上的琼脂垫上。消融后滴加M9缓冲液使线虫复苏，随后将其转移至接种了大肠杆菌的常规培养平板。复苏两天后，将经激光消融的成虫用于制备吸引实验所需的上清液。