title: Measurement of intracellular nitric oxide (NO) production in shrimp haemocytes by flow cytometry.
abstract: A flow cytometric method to measure the production of intracellular nitric oxide (NO) was adapted for use with shrimp haemocytes. We applied fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) for NO detection in haemocytes from the tiger shrimp Penaeus monodon, and used flow cytometry to quantify fluorescence intensity in individual haemocyte. The optimized protocol for intracellular NO analysis consists to incubate haemocytes with DAF-FM DA at 10 μM for 60 min to determine the mean fluorescence intensity. Result showed that NO was also produced in the untreated shrimp haemocytes. NO level in granular cells and semigranular cells were much higher than that in hyaline cells. Deﬁned by different characteristic of NO content, three subsets of haemocytes were observed. Zymosan A at dose of 10 or 100 particles per haemocyte triggered higher DAF-FM fluorescence intensity in granular and semigranular cells, than PMA that had no significant impact on all three cell types. These results indicate that granular and semigranular cells are the primary cells for NO generation. Cytochalasin B significantly inhibited the NO level induced by zymosan A. NG-Monomethyl-L-arginine (L-NMMA) and diphenylene iodonium chloride (DPI) significantly suppressed the DAF-FM fluorescence in haemocytes, but apocynin could not modulate it, indicating that the DAF-FM fluorescence was closely related to the activity of NO-synthase pathway. The NO donor sodium nitroprusside (SNP) improved the DAF-FM fluorescence in haemocytes, while the NO scavenger C-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) significantly decreased the fluorescence, demonstrating that the fluorescence intensity of DAF-FM is mainly dependent on the intracellular NO level.
authors: Key Laboratory of Ecology and Environment Science in Guangdong Higher Education, Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, School of Life Science, South China Normal University, Guangzhou 510631, People's Republic of China.
authors: Jian-An
authors: JA
authors: Xian
authors: Hui
authors: H
authors: Guo
authors: Bin
authors: B
authors: Li
authors: Yu-Tao
authors: YT
authors: Miao
authors: Jian-Min
authors: JM
authors: Ye
authors: Sheng-Peng
authors: SP
authors: Zhang
authors: Xun-Bin
authors: XB
authors: Pan
authors: Chao-Xia
authors: CX
authors: Ye
authors: An-Li
authors: AL
authors: Wang
authors: Xuan-Ming
authors: XM
authors: Hao
mesh_terms: D000818:Animals; D005434:Flow Cytometry / Q000379:methods*; D006434:Hemocytes / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D033561:Penaeidae / Q000166:cytology / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015054:Zymosan / Q000494:pharmacology
chemical_list: D009569:Nitric Oxide; D015054:Zymosan; D013755:Tetradecanoylphorbol Acetate