Pfu 2 high delity polymerase

Full-length mouse Ccr2 and Cxcr2 receptor with 3' UTR was ampli ed from total mouse RNA via reverse transcription reaction using Superscript II RT Kit (Invitrogen, Carlsbad, CA) followed by PCR using PFU II high delity polymerase (Agilent Technologies, Santa Clara, CA, USA). Resultant cDNA was inserted into pEF2-TOPO vector. Integrity of the promoter and cDNA was veri ed by direct DNA sequencing. Minimally cultured ADSC (passage 1-2) were nucleofected with pEF1-mCcr2 and pEF1-mCxcr2 plasmids, respectively, using Lonza nucleofection reaction (T-27 program, nucleofection kit V; Lonza, Cologne, Germany). Further, pool of Ccr2-and Cxcr2-expressing cells was selected with Blasticidin (0.5 mg/ml; Invitrogen) for ten days, respectively. Expression of Ccr2 and Cxcr2 in selected cells was con rmed by FACS and indirect immuno uorescence analyses. Receptor surface expression was determined by FACS using PE-conjugated antibodies. For indirect immuno uorescence, Ccr2-and Cxcr2-immunocomplexes were detected with Alexa-Fluor 488 -conjugated secondary antibodies (Invitrogen). Nuclei were counterstained with 4',6-diamidino-2-phenyl indol (DAPI; Sigma, St. Louis, MO). Immuno uorescent images were obtained on Nikon TS100F uorescent microscope (Nikon, Melville, NY, USA).

Full-length mouse Ccr2 and Cxcr2 receptor with 3' UTR was ampli ed from total mouse RNA via reverse transcription reaction using Superscript II RT Kit (Invitrogen, Carlsbad, CA) followed by PCR using PFU II high delity polymerase (Agilent Technologies, Santa Clara, CA, USA). Resultant cDNA was inserted into pEF2-TOPO vector. Integrity of the promoter and cDNA was veri ed by direct DNA sequencing. Minimally cultured ADSC (passage 1-2) were nucleofected with pEF1-mCcr2 and pEF1-mCxcr2 plasmids, respectively, using Lonza nucleofection reaction (T-27 program, nucleofection kit V; Lonza, Cologne, Germany). Further, pool of Ccr2-and Cxcr2-expressing cells was selected with Blasticidin (0.5 mg/ml; Invitrogen) for ten days, respectively. Expression of Ccr2 and Cxcr2 in selected cells was con rmed by FACS and indirect immuno uorescence analyses. Receptor surface expression was determined by FACS using PE-conjugated antibodies. For indirect immuno uorescence, Ccr2-and Cxcr2-immunocomplexes were detected with Alexa-Fluor 488 -conjugated secondary antibodies (Invitrogen). Nuclei were counterstained with 4',6-diamidino-2-phenyl indol (DAPI; Sigma, St. Louis, MO). Immuno uorescent images were obtained on Nikon TS100F uorescent microscope (Nikon, Melville, NY, USA).