|
|
Blood, Vol. 104, Issue 12, 3565-3572, December 1, 2004
Haploinsufficiency of AML1 results in a decrease in the number of LTR-HSCs while simultaneously inducing an increase in more mature progenitors
Blood Sun and Downing
104: 3565
Supplemental materials for: Sun and Downing, Vol 104 , Issue 12, 3565-3572
Fluorescence activated cell sorting (FACS) analysis. All antibodies were purchased from Pharmingen (San Jose, CA). They included: Sac-1-FITC (clone E13-161.7), c-Kit-APC (clone 2B8), and FITC- or PE-directly-conjugated lineage-specific antibodies, including: CD3 (clone 17A2), CD4 (clone RPA-T4), CD8 (clone RPT-T8), B220 (clone RA3-6B2), Ter119 (clone Ly-76), GR-1 (clone RB6-8C5) and CD11b/Mac1 (clone M1/70). Cells were first incubated with CD16/CD32 (clone 2.4G2) for 15 minutes to block Fc receptors and then with the specific antibodies in staining media (PBS containing 5% FBS and 0.01% sodium azide) on ice for an additional 30 minutes. Cells were then washed twice with staining media and analyzed using a FACSCalibur (Becton-Dickinson, San Jose, CA), and cell sorting was performed using a FACSAria. To determine the percentage of engraftment, 100 µL of blood was stained with CD45.2-FITC (against Ly5.2, clone 104) and CD45.1-PE (against Ly5.1, clone A20). Hematopoietic colony assays. Limiting dilution cobblestone area forming cell (CAFC) assay were performed using the stromal cell line FBMD-1 (a kind gift from Dr Van Zant, University of Kentucky). FBMD-1 cells were irradiated at 3000 cGy prior to the addition of hematopooietic cells. The BM cells were resuspended in murine long-term culture media (Myelocult M5300, StemCell Technologies) supplemented with fresh 1 × 10-6 M hydrocortisone, and serial dilutions were seeded at a concentration of 270 000, 90 000, 30 000, 10 000, 3000, and 1000 cells per well into 96-well plates. Typically 20 replicate wells were prepared per dilution and evaluated. Cultures were maintained at 33°C by replacing one-half of the media with fresh media weekly. At the end of 4 weeks, wells were evaluated for the presence of cobblestone cells by phase-contrast microscopy. Any well that had a colony of more than 6 cobblestone cells growing underneath the stromal cell layer was scored as positive. Cell cycle analysis. BM cells were resuspended in 1 mL of PI solution (0.05 mg/mL in 0.1% sodium citrate and 0.1% Triton X-100), and then treated with RNAse before FACS. For PY and Hoechst 33342 dual staining, cells were first incubated with 2 µg/mL Hoechst 33342 in a Hanks balanced salt solution containing 1 M HEPES, 10% FBS, 1 g/L Glucose, and 5µM reserpine for 40 minutes, and then 1.5 µg/mL PY was added and the cells incubated for an additional 20 minutes. Cells were then washed and analyzed by FACS.
Files in this Data Supplement:
- Figure S1. AML1+/+ and +/- BM have similar cellularity (JPG, 21KB)
-
BM cells were harvested from both right and left femurs and tibias and combined. Red blood cells were lysed, and the remaining white blood cells were resuspended in media and enumerated. The data is a combination of 6 independent experiments. AML1+/+ BM: 3.45 ± 0.6 × 107 cells, AML1+/- BM: 3.36 ± 0.58 × 107 cells.
- Figure S2. Schematic diagram of in vivo limiting dilution BM transplantation analysis (JPG, 81KB)
-
"Test" cells (Ly5.2+) were BM cell pools from AML1+/- or age- and sex-matched +/+ control mice. Serial dilutions of test cells were mixed with 2 - 5 × 104 Ly5.1+ radioprotective cells, and each dilution was injected into lethally irradiated Ly5.1+ syngeneic mice via tail vein. 100 µL peripheral blood was withdrawn from recipients and analyzed by FACS for the appearance of Ly5.2+ cells.
- Figure S3. AML1+/- BM cells have a normal homing capacity. (JPG, 33KB)
-
BM cells from 2-3 AML1+/- or +/+ mice were labeled in vitro with CFSE and then injected via the tail vein into lethally irradiated syngeneic recipient mice. Five hours later, cells from the BM and spleen of the recipients were harvested and analyzed by FACS for the percentage of CFSE-positive cells. Shown are results from a representative experiment using 4 × 107 labeled cells injected into 3 lethally irradiated recipient mice. Similar results were obtained when a fewer or greater number of labeled cells were used.
- Figure S4. AML1+/- BM cells from primary transplant recipients continue to show enhanced engraftment in secondary recipients. (JPG, 36KB)
-
BM cells from AML1+/+ or +/- mice (1 × 106 cells) were injected into lethally irradiated Ly5.1+ recipients (10 mice/group). The mice receiving transplants were then killed 4 months after transplantation, and Ly5.2+ cells were sorted by FACS and injected into secondary Ly5.1+ recipients. The level of donor cell engraftment in these secondary recipients was determined by FACS analysis of the BM (at 16 weeks).
|
|
