Відмінності між версіями «Gelfoam angiogenesis assay These experiments were authorized by the MD Anderson Cancer Center Institutional Animal Care and Usage Committee»
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| − | + | (IRB) of Seoul National [http://qq435579638.820.web1268.net/comment/html/?8393.html Horseradish peroxidase-conjugated (Santa Cruz Biotechnology) antibodies were used as secondary antibodies] University Hospital (SNUH) (IRB#:0902-022-271). Human T cells have been enriched from peripheral blood by utilizing RosetteSep (Stem Cell Technologies, Vancouver, Canada). Briefly, 40 ml of blood obtained from typical healthier volunteer was mixed with 2 ml of RosetteSep cocktail consisted of mouse IgG1 antibodies to human lineage antigens (CD16, CD19, CD36 and CD56) and incubated at area temperature for 30 min with gentle mixing. Immediately after dilution with an equal volume of phosphate buffered saline (PBS), T cells had been isolated by density gradient centrifugation utilizing prewarmed Ficoll-Paque (GE healthcare lifesciences, Uppsala, Sweden) at 600 g for 20 min. The interface was harvested, centrifuged at two,000 rpm for 10 min, and after that pellet was suspended to RPMI 1640 medium contained 10% FBS. Otherwise, peripheral blood was mixed with an equal volume of PBS, and loaded onto pre-warmed Ficoll-Paque. Immediately after centrifuging at 600 g for 20 min, a buffy coat containing PBMC was harvested and washed with PBS twice. The red blood cells (RBCs) were lysed with RBC lysis buffer (Sigma, St. Louis, MO, USA) inside a 37 water bath for 5 min with shaking, as well as the mononuclear cells have been washed and counted. Human T cells amongst the isolated mononuclear cells have been separated by utilizing the Pan T Cell Isolation Kit (Miltenyi Biotec, Germany) with autoMACS Pro Separator (Miltenyi Biotec, Germany) as outlined by the manufacturers' instruction. In short, determined cells were suspended with buffer and mixed with biotin-antibody cocktail (ten l/107 cells) for 5 min at 4. Following washing, cells had been mixed with anti-biotin microbeads (20 l/107 cells) for ten min at four. Washed cells were applied to the autoMACS separator, and negatively selected T cells had been counted. We confirmed greater than 95% of purified T cells were CD3+ cells by flow cytometry analysis, immediately after staining with PE-conjugated anti-CD3 antibody (eBioscience, San Diego, CA, USA). | |
| + | CD4 expression on activated T cells was reduced by stimulation with phorbol 12- myristate 13-acetate (PMA)/ionomycin reduces, but not by phytohemagglutinin (PHA) [31, 32]. Nonetheless, PHA alone can't successfully induces CD40L, but in mixture with PMA showed CD40L expression comparable to these observed using a combination of CD3 mAb and PMA [33]. Purified T cells (206/ml) have been activated by of five g/ml of PHA (Life Technologies, Grand Island, NY) for 69 hrs, then activated with 10 ng/ml of PMA (Sigma, St.Louis, MO, USA) and 1 g/ml of ionomycin for one more three hrs. Activated T cells have been analyzed by flow cytometry just after staining with FITC-conjugated anti-CD69 or CD25 antibodies (BD Pharmingen, San Diego, CA, USA). | ||
| + | MDA-MB231 cells were stained with PE-conjugated anti-human CD40 antibody (BD Pharmingen, San Diego, CA, USA), and activated T cells have been stained with FITC-conjugated antiCD25 antibody or PE-conjugated anti-CD40L antibody (BD Pharmingen, San Diego, CA, USA) for 30 min on ice. Following washing with buffer containing 0.5% bovine serum albumin (BSA) in PBS, stained cells had been analyzed by FACS Calibur (BD Bioscience, San Jose, CA, USA). To decide the Th17 differentiation by the ligation of CD40L on activated T cells with CD40 expressing MDA-MB231 cells or anti-CD40L agonistic antibody (2 g/ml), intracellular IL-17 staining with Alexa Fluor 647-conjugated anti-IL-17 antibody and surface CD4 staining with FITC-conjugated anti-CD4 antibody was performed by BD Cytofix/Cytoperm Fixation/ Permeabilization Option Kit (BD Pharmingen, San Diego, | ||
Версія за 09:21, 28 березня 2017
(IRB) of Seoul National Horseradish peroxidase-conjugated (Santa Cruz Biotechnology) antibodies were used as secondary antibodies University Hospital (SNUH) (IRB#:0902-022-271). Human T cells have been enriched from peripheral blood by utilizing RosetteSep (Stem Cell Technologies, Vancouver, Canada). Briefly, 40 ml of blood obtained from typical healthier volunteer was mixed with 2 ml of RosetteSep cocktail consisted of mouse IgG1 antibodies to human lineage antigens (CD16, CD19, CD36 and CD56) and incubated at area temperature for 30 min with gentle mixing. Immediately after dilution with an equal volume of phosphate buffered saline (PBS), T cells had been isolated by density gradient centrifugation utilizing prewarmed Ficoll-Paque (GE healthcare lifesciences, Uppsala, Sweden) at 600 g for 20 min. The interface was harvested, centrifuged at two,000 rpm for 10 min, and after that pellet was suspended to RPMI 1640 medium contained 10% FBS. Otherwise, peripheral blood was mixed with an equal volume of PBS, and loaded onto pre-warmed Ficoll-Paque. Immediately after centrifuging at 600 g for 20 min, a buffy coat containing PBMC was harvested and washed with PBS twice. The red blood cells (RBCs) were lysed with RBC lysis buffer (Sigma, St. Louis, MO, USA) inside a 37 water bath for 5 min with shaking, as well as the mononuclear cells have been washed and counted. Human T cells amongst the isolated mononuclear cells have been separated by utilizing the Pan T Cell Isolation Kit (Miltenyi Biotec, Germany) with autoMACS Pro Separator (Miltenyi Biotec, Germany) as outlined by the manufacturers' instruction. In short, determined cells were suspended with buffer and mixed with biotin-antibody cocktail (ten l/107 cells) for 5 min at 4. Following washing, cells had been mixed with anti-biotin microbeads (20 l/107 cells) for ten min at four. Washed cells were applied to the autoMACS separator, and negatively selected T cells had been counted. We confirmed greater than 95% of purified T cells were CD3+ cells by flow cytometry analysis, immediately after staining with PE-conjugated anti-CD3 antibody (eBioscience, San Diego, CA, USA). CD4 expression on activated T cells was reduced by stimulation with phorbol 12- myristate 13-acetate (PMA)/ionomycin reduces, but not by phytohemagglutinin (PHA) [31, 32]. Nonetheless, PHA alone can't successfully induces CD40L, but in mixture with PMA showed CD40L expression comparable to these observed using a combination of CD3 mAb and PMA [33]. Purified T cells (206/ml) have been activated by of five g/ml of PHA (Life Technologies, Grand Island, NY) for 69 hrs, then activated with 10 ng/ml of PMA (Sigma, St.Louis, MO, USA) and 1 g/ml of ionomycin for one more three hrs. Activated T cells have been analyzed by flow cytometry just after staining with FITC-conjugated anti-CD69 or CD25 antibodies (BD Pharmingen, San Diego, CA, USA). MDA-MB231 cells were stained with PE-conjugated anti-human CD40 antibody (BD Pharmingen, San Diego, CA, USA), and activated T cells have been stained with FITC-conjugated antiCD25 antibody or PE-conjugated anti-CD40L antibody (BD Pharmingen, San Diego, CA, USA) for 30 min on ice. Following washing with buffer containing 0.5% bovine serum albumin (BSA) in PBS, stained cells had been analyzed by FACS Calibur (BD Bioscience, San Jose, CA, USA). To decide the Th17 differentiation by the ligation of CD40L on activated T cells with CD40 expressing MDA-MB231 cells or anti-CD40L agonistic antibody (2 g/ml), intracellular IL-17 staining with Alexa Fluor 647-conjugated anti-IL-17 antibody and surface CD4 staining with FITC-conjugated anti-CD4 antibody was performed by BD Cytofix/Cytoperm Fixation/ Permeabilization Option Kit (BD Pharmingen, San Diego,
