Hemocytes are immune cells in the hemolymph of invertebrates that play multiple roles in response to stressors; hemocyte mortality can thus serve as an indicator of overall animal health. However, previous research has often analyzed hemolymph samples pooled from several individuals, which precludes tracking individual responses to stressors over time. The ability to track individuals is important, however, because large inter-individual variation in response to stressors can confound the interpretation of pooled samples. Here, we describe protocols for analysis of inter- and intra-individual variability in hemocyte mortality across repeated hemolymph samples of California mussels, Mytilus californianus, free from typical abiotic stressors. To assess individual variability in hemocyte mortality with serial sampling, we created four groups of 15 mussels each that were repeatedly sampled four times: at baseline (time zero) and three subsequent times separated by either 24, 48, 72, or 168 h. Hemocyte mortality was assessed by fluorescence-activated cell sorting (FACS) of cells stained with propidium iodide. Our study demonstrates that hemolymph can be repeatedly sampled from individual mussels without mortality; however, there is substantial inter- and intra-individual variability in hemocyte mortality through time that is partially dependent on the sampling interval. Across repeated samples, individual mussels' hemocyte mortality had, on average, a range of ∼6% and a standard deviation of ∼3%, which was minimized with sampling periods ≥72 h apart. Due to this intra-individual variability, obtaining ≥2 samples from a specimen will more accurately establish an individual's baseline. Pooled-sample means were similar to individual-sample means; however, pooled samples masked the individual variation in each group. Overall, these data lay the foundation for future work exploring individual mussels' temporal responses to various stressors on a cellular level.

血细胞是无脊椎动物血淋巴中的免疫细胞，对应激源起多种作用。因此，血细胞死亡率可以作为总体动物健康的指标。但是，先前的研究经常分析从几个人收集的血淋巴样本，从而无法跟踪随时间推移对应激源的个体反应。但是，跟踪个体的能力很重要，因为对压力源的个体间较大差异会混淆合并样本的解释。在这里，我们描述了加利福尼亚贻贝，Mytilus californianus重复的血淋巴样品中血细胞死亡率的个体间和个体内变异性分析协议，不含典型的非生物应激源。为了通过连续采样评估血细胞死亡率的个体差异性，我们创建了四组，每组15个贻贝，每组重复采样四次：在基线（零时），随后的三个时间分别间隔24、48、72或168小时。通过用碘化丙啶染色的细胞的荧光激活细胞分选（FACS）评估血细胞死亡率。我们的研究表明，可以从单个贻贝中反复采集血淋巴而不会导致死亡；但是，随着时间的流逝，血细胞死亡率在个体之间和个体之间存在很大的差异，部分取决于采样间隔。在重复的样本中，贻贝的单个血细胞死亡率平均约为6％，标准偏差约为3％，相距≥72小时的采样时间最小。由于这种个体内部差异性，从样本中获取≥2个样本将更准确地建立个体的基线。汇总样本均值类似于单个样本均值；但是，合并样本掩盖了每组中的个体差异。总体而言，这些数据为将来的工作奠定了基础，以探索单个贻贝在细胞水平上对各种压力源的时间响应。