Reagents required for blood collection, fixation, and staining included: a heparin-based anticoagulant solution, buffer solution, methanol fixative, anti-CD71-FITC, anti-CD61-PE, RNase, and propidium iodide. These reagents are all components of the commercially available kit: Mouse MicroFlowPLUS® Kit (Litron Laboratories, Rochester, NY). The instrument calibration standard, fixed Plasmodium berghei-infected mouse erythrocytes (“malaria biostandard”), was also provided in these kits.
Female C57BL/6N mice were purchased from the National Cancer Institute. Mice were allowed to acclimate for at least one week prior to treatment. Mice were handled in accordance with the standards established by the U.S. Animal Welfare Acts set forth in the National Institutes of Health (NIH) guidelines. All procedures were reviewed and approved by the University of Rochester’s Committee on Animal Resources. Purina Mills Rodent Chow 5001 and water were available ad libitum.
The age of mice at the time of treatment was between 8 and 9 weeks. For three independent dose-response experiments, treatments were performed with groups of 5 mice, and doses ranged from 0.125 to 3.0 Gy. For the time-course experiment, each dose and each time point consisted of separate groups of 4 mice, and doses ranged from 1.0 to 2.0 Gy. Total body irradiation was performed with a single dose of 137Cs gamma-rays. The dose rate was approximately 2 Gy per minute. Control animals were sham-irradiated. After irradiation, the mice were maintained in groups of 5 or fewer animals per cage in pathogen-free rooms.
2.4 Blood Collection and Fixation
Blood collection occurred via an incision to the tail vein. To facilitate bleeding, mice were warmed briefly under a heat lamp. Approximately 100 μl of free-flowing blood was collected into tubes containing 350 μl heparin solution. Heparinized blood was maintained at room temperature until fixation occurred (within 4 hours). For each of three dose-response experiments, blood was collected 43 hrs post-exposure. For the time-course experiment, blood was collected at 12 hr intervals between 43 and 115 hrs post-exposure.
Heparinized blood samples were fixed with ultracold methanol according to Mouse MicroFlowPLUS Kit instructions. These fixed blood specimens were returned to the ultracold freezer for storage until flow cytometric analyses were performed as described below.
2.5 Staining and Flow Cytometric Analyses
Methanol-fixed blood samples were washed and labeled for flow cytometric analysis according to procedures described in the Mouse MicroFlowPLUS Kit manual (vP4.3M). Samples were analyzed by a flow cytometer equipped with a 488-nm laser (FACSCalibur, Becton Dickinson, San Jose, CA). Anti-CD71-FITC, anti-CD61-PE, and propidium iodide fluorescence signals were detected in the FL1, FL2, and FL3 channels, respectively (stock filter sets). The gating logic used to ensure that quantitative analyses of erythrocyte subpopulations where not contaminated by other cellular or noncellular events has been described previously [7
Calibration of the flow cytometer was accomplished by staining malaria-infected erythrocytes in parallel with test samples on each day of analysis [12
]. By adjusting voltages applied to the photomultiplier tube, it was possible to standardize the propidium iodide-associated fluorescence intensity exhibited by erythrocytes housing a single parasite (i.e., an MN-like event). In this manner, analysis regions were consistent between experiments.
Data were acquired with CellQuest software (v3.3, BD-Immunocytometry Systems, San Jose, CA). The stop mode was set so that 20,000 CD71-expressing RETs were analyzed per blood sample. As shown in , four erythrocyte subpopulations were enumerated, thereby providing the means to calculate %RET, an index of erythropoiesis function, and %MN-RET, an index of recent chromosomal damage.
Figure 1 Representative 0, 0.5 and 1.0 Gy specimens (panels A, B, and C, respectively) illustrate fluorescent staining characteristics of four erythrocyte subpopulations: lower left quadrant = mature erythrocytes; lower right = micronucleated mature erythrocytes; (more ...)
All statistical analyses were conducted using SAS version 9.1 (SAS Institute, Cary, NC, USA). All tests were two-sided and conducted at the 5% level of significance.
2.6.1 Dose-Response Experiments
Five mice were randomly allocated to each experimental block—one block being defined by a dose of irradiation and an independent experiment. A single flow cytometric analysis was performed per mouse, and observations were considered independent across mice. Our primary endpoints were %MN-RETs and %RETs. Basic descriptive statistics, including the means, medians and standard deviations, were computed for each block. The significance of the dose-response curve was assessed for each endpoint using the nonparametric Kruskal-and-Wallis test. The results were confirmed by (parametric) analyses of variance (ANOVA), where formal hypotheses testing was conducted using F
-tests. Nonparametric Wilcoxon rank-sum tests were used for pairwise comparisons of dose groups. Linear regression analyses including dose as a continuous covariate were conducted to assess the decrease in %MN-RETs observed for doses > 1 Gy. Our hypothesis was tested using a F
-test. Wherever needed, the results of independent analyses were combined using Fisher’s method for combining p
2.6.2 Time-Course Experiments
Four mice were randomly allocated to each of the 21 experimental blocks (each block being defined by a dose and a time point). The primary endpoints were also %MN-RETs and %RETs. The nonparametric Kruskal-and-Wallis test was used to assess the existence of differences between the medians of %RETs across time points. One such test was conducted for each dose of irradiation. The effect of dose on %RETs was also assessed using the same test. This was done for each time point separately. Similarly analyses were carried out to investigate %MN-RETs, and its relationship with time post-irradiation and with the dose of gamma-rays. The results of these primary statistical analyses were confirmed using parametric ANOVA, where formal hypotheses testing was carried out using F-tests.