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通过对流式细胞术(FCM)检测虾类血细胞总数(THC)的条件和方法进行优化,为虾类血细胞学研究提供快捷、准确的测定方法。2012年7-9月,应用SYBR Green I作为荧光染料标记完整血细胞,设置3个不同染料浓度(1×、10×和100×),测定不同孵育时间下染色细胞比例的变化。结果显示,染料终浓度为10×时染色效果最佳,其最佳孵育时间为60 min;应用建立的FCM方法和显微计数方法测定10尾凡纳滨对虾(Litopenaeus vannamei)的THC,平均值分别为(16.68±1.57)×106个/m L和(15.09±1.76)×106个/m L,2种方法测定结果的相关性极显著(R2=0.8064,P<0.01)。凡纳滨对虾经不同浓度(0.5 mg/L和5.0 mg/L)的Cd2+胁迫,利用建立的FCM方法测定Cd2+胁迫下对虾THC的变化。结果显示,0.5 mg/L和5.0 mg/L Cd2+胁迫下,对虾THC随着胁迫时间的延长不断下降,胁迫48 h时分别下降至对照组的78.7%和64.7%,可见Cd2+胁迫对虾类血细胞产生毒性,抑制了血细胞活性,表明该方法适用于虾类的血细胞学研究。
Optimization of the conditions and methods for the detection of the total shrimp blood cells (THC) by flow cytometry (FCM) provides a fast and accurate method for the determination of shrimp blood cells. From July to September in 2012, whole blood cells were labeled with SYBR Green I as a fluorescent dye. Three different dye concentrations (1 ×, 10 × and 100 ×) were set up to determine the changes of the proportion of stained cells at different incubation times. The results showed that the optimal dyeing time was 10 × and the optimal incubation time was 60 min. The FCM method and microscopic counting method were used to determine the THC of 10 Litopenaeus vannamei (16.68 ± 1.57) × 106 cells / m L and (15.09 ± 1.76) × 106 cells / m L, respectively. The correlation between the two methods was significant (R2 = 0.8064, P <0.01). Litopenaeus vannamei was treated with Cd2 + at different concentrations (0.5 mg / L and 5.0 mg / L), and the changes of THC in shrimp were determined by FCM. The results showed that under 0.5 mg / L and 5.0 mg / L Cd2 + stress, the THC of shrimp decreased with the prolongation of stress time and decreased to 78.7% and 64.7% of the control at 48 h, indicating that the Cd2 + Toxicity, inhibition of blood cell activity, indicating that the method is suitable for shrimp blood cells.