Fig 1.
GFP expression in reporter macrophages.
GFP and FLUC expressing reporter macrophages were produced by transducing mouse bone marrow macrophages with lentivirus vector in the presence of cyclosporine, DEAE-dextran or polybrene. The 24-hour virus infection was done either once or repeated after a 24-hour rest period (single vs. double infection). a) Fluorescence microscopy images of the GFP positive macrophages in the indicated treatment groups three days after the second infection. b) Representative scatter blots of GFP positive reporter macrophage in flow cytometry. c) Percentage of GFP positive macrophages in the indicated treatment groups as determined by flow cytometry. Cy–Cyclosporine; De–DEAE-dextran; Po–Polybrene; D–Double infection; S–Single infection; *** = p > 0.001 cyclosporine double infection vs. dextran and polybrene single and double infection; * = p > 0.05 cyclosporine double vs. single infection; + = p > 0.05 cyclosporine single infection vs. dextran and polybrene single and double infection.
Fig 2.
FLUC expression in reporter macrophages.
GFP and FLUC expressing reporter macrophages were produced by transducing mouse bone marrow macrophages with lentivirus vector in the presence of cyclosporine, DEAE-dextran or polybrene. The 24-hour virus infection was done either once or repeated after a 24-hour rest period (single vs. double infection). a) Reporter macrophages from the indicated treatment groups were seeded to 24-well plate at standardized density three days after the second infection. D-luciferin was added to the wells and immediately after luminescence signal emanating from each well imaged using IVIS 100 system and quantified with Living Image software. b) Luciferase activity in indicated double infected reporter macrophage lysates as determined by Luciferase Assay System kit and luminometer. c) Person correlation between the amount of GFP positive cells and normalized FLUC activity in macrophages infected once or twice in the presence of cyclosporine. D–Double infection; S–Single infection; Cy–Cyclosporine; De–DEAE-dextran; Po–Polybrene. * = p > 0.05 cyclosporine double infection vs. polybrene double infection.
Table 1.
Reporter macrophage viability.
GFP and FLUC expressing reporter macrophages were produced by transducing mouse bone marrow macrophages with lentivirus vector in the presence of cyclosporine, DEAE-dextran or polybrene. The 24-hour virus infection was done either once or repeated after a 24-hour rest period (single vs. double infection). The percentage of viable cells was assessed three days after the second infection by flow cytometry and propidium iodide staining.
Fig 3.
Reporter macrophage polarization.
Lentivirus and cyclosporine transduced reporter macrophages and non-transduced control macrophages were polarized into M1 and M2 phenotypes three days after the virus infection and the expression of M1-related genes TNF-α, iNOS and IRF5 as well as M2-realted genes CD206, Arg1 and IRF4 was determined by qRT PCR. Results are expressed as common logarithm-transformed fold change (fc) to corresponding non-polarized M0 macrophages.
Fig 4.
GFP and FLUC expression in polarized reporter macrophages.
Lentivirus and cyclosporine transduced reporter macrophages were polarized into M1 and M2 phenotypes or left untreated (M0 macrophages) and the expression of GFP and FLUC determined 48 hours after the end of the polarization treatment. a) Fluorescence microscopy images of the GFP positive macrophages in the indicated treatment groups. b) Ratio of GFP positive macrophages in the indicated treatment groups compared to non-polarized M0 reporter macrophages as determined by flow cytometry c) FLUC activity in indicated treatments groups compared to the non-polarized M0 reporter macrophages as determined by Luciferase Assay System kit and luminometer.
Fig 5.
Reporter macrophages in in vivo bioluminescence imaging.
Lentivirus and cyclosporine transduced reporter macrophages where injected into the tail vein of mouse model of biomaterial induced local inflammation and osteolysis. The cell trafficking was followed by bioluminescence imaging. Luminescence emanating from the local area of inflammation in the right distal femur as well as left distal femur serving as a control was determined from images obtained every other day up to 20 days post injection. a) Bioluminescence images showing the accumulation of reporter cells to the right distal femur starting on the day 2 after the systemic injection of the reporter cells and persisting over the 20 day imaging period. b) XY-blot showing the total flux from regions of interest over the right and left distal femurs. * = p > 0.05; ** = p > 0.01, *** = p > 0.001.
Fig 6.
Detection of reporter macrophages from tissue sections.
Lentivirus and cyclosporine transduced reporter macrophages where injected into the tail vein of mouse model of biomaterial induced local inflammation and osteolysis. Reporter cell accumulation to the right distal femur was confirmed by bioluminescence imaging. After 21 days the right and left femur were collected, processed into frozen sections and the reporter macrophages detected by FLUC immunostaining. a) Low power micrographs showing the transverse sections of distal metaphyseal region of left and right femur. On the right side the channel left behind by the titanium rod is evident (asterisk). b) High power micrographs showing several aggregations of FLUC positive cells (arrow heads) in the bone marrow surrounding the rod channel on the right side while only few FLUC positive cells are detected in the bone marrow of the left femur. c) GFP-FLUC transduced and non-transduced RAW 264.7 cells stained for FLUC served as additional positive and negative immunostaining controls. Scale bars 200μm.