"10" . "Vnitrobun\u011B\u010Dn\u00E1 detekce cytokin\u016F pr\u016Ftokovou cytometri\u00ED u prasete: fixace, permeabilizace a povrchov\u00E9 zna\u010Den\u00ED."@cs . "Intracellular cytokine detection by flow cytometry in pig: fixation, permeabilization and cell surface staining."@en . "[3ACEC2EECEAE]" . . "427327" . "5"^^ . . . . . . "NL - Nizozemsko" . "12"^^ . "Faldyna, Martin" . . . "3"^^ . . "327" . "Cytokine detection; Flow cytometry; Pig; Fixation and permeabilization; Cell surface staining"@en . "\u0160t\u011Bp\u00E1nov\u00E1, Hana" . "Zeln\u00ED\u010Dkov\u00E1, Petra" . "RIV/62157124:16170/07:00001432" . . "Journal of Immunological Methods" . "Kov\u00E1\u0159\u016F, Franti\u0161ek" . "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 T\u00FCk4). While anti-CD8 (clone MIL-12) must be used for staining of unfixed cells, the other ant"@en . "Intracellular cytokine detection by flow cytometry in pig: fixation, permeabilization and cell surface staining."@en . . . . "P(GA524/05/0267), Z(MSM6215712403)" . . "Intracellular cytokine detection by flow cytometry in pig: fixation, permeabilization and cell surface staining." . "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 T\u00FCk4). While anti-CD8 (clone MIL-12) must be used for staining of unfixed cells, the other ant" . "RIV/62157124:16170/07:00001432!RIV08-GA0-16170___" . . "Intracellular cytokine detection by flow cytometry in pig: fixation, permeabilization and cell surface staining." . . "18-29" . "0022-1759" . . "Vnitrobun\u011B\u010Dn\u00E1 detekce cytokin\u016F pr\u016Ftokovou cytometri\u00ED u prasete: fixace, permeabilizace a povrchov\u00E9 zna\u010Den\u00ED."@cs . "16170" . . . . "Ondr\u00E1\u010Dek, Jaroslav" . . "Pr\u016Ftokov\u00E1 cytometrie spojen\u00E1 s vnitrobun\u011B\u010Dn\u00FDm zna\u010Den\u00EDm je metodou detekce cytokin\u016F, kter\u00E1 umo\u017E\u0148uje simult\u00E1nn\u00ED detekci intracelul\u00E1rn\u011B deponovan\u00FDch cytokin\u016F spole\u010Dn\u011B s povrchov\u00FDm zna\u010Den\u00EDm. Tato d\u016Fle\u017Eit\u00E1 metoda nen\u00ED dostate\u010Dn\u011B pou\u017E\u00EDvan\u00E1 \u010D\u00E1ste\u010Dn\u011B proto\u017Ee nejsou dostupn\u00E9 metodick\u00E9 protokoly modifikuj\u00EDc\u00ED protokoly pro zna\u010Den\u00ED u lid\u00ED. C\u00EDlem t\u00E9to studie bylo naj\u00EDt nejlep\u0161\u00ED proceduru pro fixaci a permeabilizaci prase\u010D\u00EDch krevn\u00EDch leukocyt\u016F a simult\u00E1nn\u00ED povrchov\u00E9 zna\u010Den\u00ED. Permeabilizace komer\u010Dn\u00EDmi kity d\u00E1valy lep\u0161\u00ED v\u00FDsledky ve v\u011Bt\u0161in\u011B sledovan\u00FDch parametr\u016F ve srovn\u00E1n\u00ED s kombinac\u00ED r\u016Fzn\u00FDch koncentrac\u00ED paraformaldehydu a saponinu. Mezi testovan\u00FDmi komer\u010Dn\u00EDmi kity byly nejlep\u0161\u00ED v\u00FDsledky dosa\u017Eeny p\u0159i pou\u017Eit\u00ED kitu IntraStain. Povrchov\u00E9 znaky byly detekovateln\u00E9 na bu\u0148k\u00E1ch stimulovan\u00FDch k produkci cytokin\u016F protil\u00E1tkou proti CD14 (klon MIL-2), proti SWC3, CD4 aCD8 krom\u011B protil\u00E1tky proti CD14 klon T\u00FCk4. Zat\u00EDmco protil\u00E1tka proti CD8 klon MIL-12 mus\u00ED b\u00FDt pou\u017Eita na nefixovan\u00FDch bu\u0148k\u00E1ch, ostatn\u00ED protil\u00E1tky mohou b\u00FDt pou\u017Eity"@cs .