To study the effects of radiation on activation of repair genes and induction of DNA damage, cells were analyzed for gamma-H2AX staining by flow cytometry and immunofluorescence. Gamma-H2AX marks sites of double-strand breaks. CSCs or MSCs grown either in 6 well plates or in 2 well chamber slides were exposed to radiation at BNL. For immunofluorescence, 15 minutes, 1, 2, 6, 12, 18, and 24 hours after, cells were fixed in cold acetone: methanol (1:1). Media was collected at each time for cytokine analysis later. The fixed cells were analyzed for DAPI (red-nucleus) and immunofluorescence staining with gamma-H2AX antibody (green). For flow cytometry, cells were trypsinized and stained with FITC conjugated gamma-H2AX antibody and propidium iodide (PI) at 15 minutes, 1, 2, 6, 12, and 18 hours after radiation and analyzed. HFH74 cells showed a 2-4 fold increase in the staining up to 2 hours following 100 cGy of proton irradiation. At later time points, this increase was lost. Following HZE ion irradiation however, an increase in gamma-H2AX was observed at 6 hours or 18 hours. Significant induction of gamma-H2AX was not observed in NHMSC cells following proton irradiation; however, an increase in gamma-H2AX was observed at 6 hours or 18 hours following Iron irradiation. In the case of Iron irradiation, a significant increase in cell death was observed up to two hours following 20 cGy exposure in both the cell lines. However, it appears that cells were able to recover from this initial damage. Since flow cytometry provides an average number of positive events in a cell population, investigators are currently analyzing gamma-H2AX staining using immunofluorescence.
One potential effect of space flight-relevant radiation may be to induce aging of stem cells and therefore investigators compared the CFU-F formation potential of cells after radiation exposure. In NHMSC cells following Iron irradiation (10cGy), a surviving fraction (SF) of 0.43 ± 0.0 was observed. Following continuous gamma irradiation for 12 hours, a SF of 0.91 ± 0.1 at 50mR/h and 0.8 ± 0.1at 100mR/h was observed. HFH74 cells showed a plating efficiency of only 7% even in matrigel coated plates, therefore the growth of the cells with and without radiation was followed in real time using cell electronic sensing system (ACEA Biosciences). As expected, with all types of irradiation in HFH74 cells increased growth delay was observed with increasing radiation dose. However, in NHMSC cells, except following proton irradiation, significant differences in growth were not observed with the increasing doses of radiation.
No datasets exist for this study. A final report was archived.
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