The aim of this work was to evaluate ontogeny of reactive nitrogen species (RNS) production by peripheral blood phagocytes in pig. Pig fetuses (55 and 92 days of gestation) and postnatal piglets (1, 3, 8, 17, 31 and 41 days after birth) were used. RNS production was measured by fluorescent probes diaminofluorescein-diacetate (DAF-FMDA) and dichloro-fluorescein-diacetate (H2DCFDA). Levels of nitration of cell proteins were established by immunofluorescent detection of nitrotyrosine. Levels of plasma nitrites/nitrates were detected spectrophotometrically by Griess reaction. Nitric oxide production measured by DAF-FMDA in neutrophils decreased during postnatal life. Spontaneous RNS measured by H2DCFDA decreased from 55th day of gestation to the 41st day of life. Phorbol-12-myristate-13-acetate activated production decreased during postnatal life. Production of NO measured by DAF-FMDA in macrophages decreased from the first to 41st day after birth. RNS production measured by H2DCFDA in monocytes did not show any significant changes during ontogeny. The level of nitrotyrosine significantly decreased from the third to 17th day. Levels of plasma nitrites/nitrates gradually decreased from the 55th day of gestation to the 41st day after birth. A temporary increase in all parameters occurred after weaning, but without any significance. In conclusion, RNS production has a decreasing trend during ontogeny and is transiently upregulated after weaning.
Intracellular flow cytometry is a method of cytokine detection that allows simultaneous detection of intracellular cytokines and cell surface markers. This important method is not extensively used in pigs, in particular due to the inaccessibility of proper methodological protocols modifying comprehensive human protocols. The aim of this study was to find the best procedure for fixation and permeabilization of porcine blood leukocytes and simultaneous cell surface staining. Permeabilization with commercial kits gave better results in most of the chosen parameters compared with combinations of different concentrations of paraformaldehyde and saponin. Among the commercial kits tested, the best results were obtained with the IntraStain kit. Cell surface markers were detected on cells stimulated for cytokine production by antibodies anti-CD14 (clone MIL-2), anti-SWC3, anti-CD4 and anti-CD8 except anti-CD14 (clone Tuk4). While anti-CD8 (clone MIL-12) must be used for staining of unfixed cells, the other antibodies recognize fixed and/or permeabilized cells. Moreover, anti-SWC3 and anti-CD14 (clone MIL-2) antibodies can stain cells during the permeabilization step. These modifications of the cell surface staining protocol allow the researcher to speed up the procedure of intracellular cytokine staining or to combine cell surface staining and intracellular cytokine staining. The present study can serve as a particular protocol of intracellular cytokine detection and as a suggestion for optimization of the fixation, permeabilization and cell surface staining procedure in any laboratory.
- MeSH
- barvení a značení MeSH
- buněčná membrána metabolismus MeSH
- cytokiny analýza MeSH
- cytoplazma metabolismus MeSH
- financování organizované MeSH
- fixace tkání MeSH
- fixativa MeSH
- fluorescenční protilátková technika MeSH
- leukocyty mononukleární metabolismus MeSH
- lidé MeSH
- prasata MeSH
- průtoková cytometrie metody MeSH
- senzitivita a specificita MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
Učebnice pro lékařské fakulty
4. přeprac. vyd 292 s. : il.
- Klíčová slova
- nemoci infekční, učebnice vysokoškolské,
- NLK Obory
- infekční lékařství
- NLK Publikační typ
- učebnice vysokých škol
