Technical Reference #1625

1625.

In Vitro Generation of Murine Myeloid Dendritic Cells from CD34-positive Precursors Tom-Li Stephen; Fabian Harms; Mario Fabri; Eva Flenner; Martina Bessler; Helena Hafke; Sonja Meemboor; Christoph Kalka; Wiltrud Kalka-Moll, University of Cologne, Cell Biology International, 33(1625), (2009)

Abstract:
Dendritic cells (DCs) link the innate and adaptive immune system. Currently murine DCs for cell biology investigations are developed fromMHC class II-negative bone marrow (BM) precursor cells non-depleted BM cells or BM monocytes in the presence of gr

Keywords:
Murine dendritic cells; CD34; Bone marrow; Antigen presentation

Materials & Methods:
2.8. Fluorescence microscopy Cells transfected adenovirally with Rab5-EGFP Rab7-EGFP and Rab11b-EGFP were imaged by inverted fluorescent microscopy on an Olympus IX81 microscope as previously described (Chow et al. 2002; Stephen et al. 2007). Temperature control at 37.8 C was achieved with a heating dish. Acquisition was performed using AnalySIS Imaging System software (Olympus). Cells transfected retrovirally with MHCII-GFP (I-Ea-EGFP) were imaged by live confocal microscopy as previously described (Chow et al. 2002; Stephen et al. 2007). In brief cells were plated for 30 min on poly-D-lysine-pre-coated number 1.5 coverslips attached to 35-mm dishes (MatTek) and fresh medium was added. Cells were washed before and after ovalbumin- Alexa 594 treatment three times in ice-cold medium. Confocal microscopy was done on a PerkinElmer UltraView LCI spinning disc system equipped with a suitable multi-band beamsplitter and a MellesGriot Omnichrome 643-RYB-A02 ArKr gas laser providing 488-nm and 568-nm lines for excitation. A Nikon Plan Fluor 3100 1.3NA oil immersion objective and 525/50 and 607/45 emitter filters were used for GFP and Alexa 594 respectively. On confocal microscopy environmental condition was controlled by a custom incubator (EMBL GP 168) that provides a 37.8 C and 5% CO2 atmosphere. Images were exported to TIFF images processed using Adobe Photoshop version 6.0.

Microscopic Technique
Fluorescence Microscopy

Cell Type(s)
CD34+ T