Task Progress:
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A549 (human lung fibroblasts) cells, used for radiation, microgravity, and combined effects (10 Gy Gamma + microgravity) experiments, were cultured 3 days prior to treatment. 500 x 10(3) A549 cells were cultured per T-25 for radiation experiments using standard cell culturing techniques and media (10% FBS containing DMEM with high glucose and pyruvate). For microgravity (n=2) and combined effects (n=2) experiments, 350 x 10(3) A549 cells were cultured per Synthecon cell rotator with Cytodex(3) microcarriers. A459 cells were exposed to Gamma at Duke University using the J. L. Shepherd Mark I Model 68A 137Cs Gamma irradiator (8 Gy (n=12) and 10 Gy (n=8)). Each experimental group was treated at the same time and harvested for 2-day and 5-day post-radiation assessment using flow cytometry A459 cells irradiated at Brookhaven National Laboratory (BNL) and harvested at 1-day post-radiation for flow cytometry (150 cGy GCR5-ion samples (n=4)). For all experiments, untreated controls were cultured and harvested identically to their treated counterparts. Indirect flow cytometry assay was developed using the J2 primary antibody, mouse monoclonal anti-double stranded (ds) RNA antibody from Jena Bioscience GmbH, and secondary antibody, Alexa 488 conjugated anti-mouse IgG polyclonal antibody from BioLegend. All cells were fixed and permeabilized using eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set from Invitrogen. 10,000 events were recorded per sample and FlowJo v10.8 (Becton, Dickinson and Company) was used to gate singlet populations for average median fluorescence intensity (aMFI) of detected dsRNA.
For individual effects treated samples at 2-days post-radiation, only 8 Gy Gamma showed a significant elevation in aMFI—a 1.30-fold increase (p=0.0175). Combined 10 Gy Gamma and microgravity effects at the 2-day post-radiation timepoint showed a 1.73-fold increase (p=0.0002) in aMFI compared to normal gravity controls and a 1.94-fold increase (p<0.0001) in aMFI compared to 10 Gy Gamma only treated samples. 5-days post-radiation results, however, showed statistical significance across all experimental treatment groups; 8 Gy Gamma, 10 Gy Gamma, and microgravity treated samples showing a 1.33-fold increase (p=0.0155), 2.13-fold increase (p=0.0004), and 2.41-fold increase (p=0.0004) in aMFI, respectively. Combined effects of 10 Gy Gamma and microgravity at the 5-day post-radiation timepoint showed a 4.72-fold (p<0.0001) increase in aMFI compared to normal gravity controls and a 1.96-fold (p=0.0215) increase in aMFI compared to microgravity only treated samples. 1-day post-radiation samples irradiated with GCR5-ion displayed a 1.21-fold increase in aMFI although it was not statistically significant (p=0.47).
This study was significantly affected by Covid 19 shutdown. From another study we have developed an extensive tissue repository of mice that have been exposed to various type of radiation including 5 ion GCR sim. Now that we have developed the indirect immunofluorescence assay with the J2 antibody we plan to evaluate some of the tissue in this repository.
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