Cells were pelleted and resuspended in 3 ml DMEM- 10% FBS-100 ��g DNaseI-10 ��M Y27632 (Sigma, St. Louis, MO). Verification that the cell suspension was dissociated into single cells was performed by placing 200 ��l of the preparation into a well of a 96-well plate and observing the cells under a fluorescent microscope EMD 1214063 as well as under bright-field optics. Quantification of cells expressing distinct Sox9-EGFP levels (High, Low, and Sublow or Negative) was performed by using a Cyan flow cytometer, Summit v4.3 software, and parameters previously described (21). Propidium iodide staining (Sigma, St. Louis, MO) was used to gate out dead cells. Immune cells were excluded by forward-side scatter gating and doublets were discriminated by use of both forward scatter and side scatter height-width plots.

For each flow cytometry run, the proportion of each cell population at each time point after radiation was compared with the proportion of the respective cell population in nonirradiated control mice studied in the same run. Tissue Harvest for FACS of Different Sox9-EGFP-Expressing Cell Populations These studies focused on nonirradiated mice and mice at 5 days after irradiation when histology revealed maximum crypt regeneration/cell proliferation. FACS was used to isolate cells for testing in the ISC/organoid culture system and for gene microarray. For these analyses, 6- to 10-wk-old nonirradiated Sox9-EGFP mice or Sox9-EGFP mice at day 5 after abdominal radiation were euthanized with a lethal dose of Nembutal and the entire jejunum was placed on ice and processed for IEC isolation and dissociation into single cells as described above.

Prior to FACS, the dissociated cells were sequentially passed through 100-��m, 70-��m, and 30-��m filters. The cells were pelleted and placed in 3 ml Advanced DMEM F12�C10% FBS-100 ��g DNaseI-10 ��M Y27632 (Sigma). Sorting of Sox9-EGFP High, Low, Sublow, and Negative cells was performed by using a MoFlo XDP FACS machine (Dako/Cytomation, Carpinteria, CA) and Summit v4.3 software. Dead and immune cells were excluded by forward-side scatter gating and doublets were discriminated by using both forward scatter and side scatter height-width plots. For each sort of a preparation of dissociated cells from an irradiated mouse, gates delimiting the distinct Sox9-EGFP populations were defined by use of a nonirradiated control littermate sorted in the same run.

Postsort analysis indicated that the different Sox9-EGFP cell populations were correctly isolated on the basis of EGFP status (Fig. 1A). In addition, postsort efficiencies were assessed by flow cytometry after each run for each sample to verify Carfilzomib minimal contamination by other Sox9-EGFP cell types (Fig. 1B). Results demonstrated high postsort efficiencies and negligible cross-contamination of Sox9-EGFP High, Low, Sublow, and Negative cells with other cell populations (Fig. 1B). Fig. 1.