Pfu 2 high fidelity polymerase

Full-length mouse Ccr2 and Cxcr2 receptor with 3′ UTR was amplified from total mouse RNA via reverse transcription reaction using Superscript II RT Kit (Invitrogen, Carlsbad, CA, USA) followed by PCR using PFU II high fidelity polymerase (Agilent Technologies, Santa Clara, CA, USA). Resultant cDNA was inserted into pEF2-TOPO vector. Integrity of the promoter and cDNA was verified 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 10 days, respectively. Expression of Ccr2 and Cxcr2 in selected cells was confirmed by FACS and indirect immunofluorescence analyses. Receptor surface expression was determined by FACS using PE-conjugated antibodies. For indirect immunofluorescence, Ccr2- and Cxcr2-immunocomplexes were detected with Alexa-Fluor⁴⁸⁸-conjugated secondary antibodies (Invitrogen). Nuclei were counterstained with 4′,6-diamidino-2-phenyl indol (DAPI; Sigma, St. Louis, MO, USA). Immunofluorescent images were obtained on Nikon TS100F fluorescent microscope (Nikon, Melville, NY, USA).

Full-length mouse Cxcr2 receptor with 3′ UTR was amplified from total mouse RNA via reverse transcription reaction using the Superscript II RT Kit (Invitrogen, Carlsbad, CA, USA) followed by PCR using PFU II high fidelity polymerase (Agilent Technologies, Santa Clara, CA, USA). Resultant cDNA was inserted into pEF2-TOPO vector. Integrity of the promoter and cDNA was verified by direct DNA sequencing. Minimally cultured mADSC (passages 1–2) were nucleofected with resultant plasmid (pEF1-mCxcr2) using Lonza nucleofection reaction (T-27 program, nucleofection kit V; Lonza, Cologne, Germany). Further, a pool of CXCR2-expressing cells was selected with Blasticidin (0.5 mg/ml; Invitrogen) for 10 days. Expression of CXCR2 in selected cells was confirmed by FACS and indirect immunofluorescence analyses. Surface expression of CXCR2 was determined by FACS using PE-conjugated antibodies as already described. For indirect immunofluorescence, CXCR2 immunocomplexes were detected with Alexa-Fluor⁴⁸⁸-conjugated secondary antibodies (Invitrogen). Nuclei were counterstained with 4′,6-diamidino-2-phenyl indol (DAPI; Sigma). Immunofluorescent images were obtained on a Nikon TS100F fluorescent microscope (Nikon, Melville, NY, USA).