Competitive transplantations were performed using donor BM cells (2.5 × 104, 5 × 104, or 1 × 105) from young and old C57BL/6 mice (CD45.1CD45.2+) isolated as described in Figure 2A (23). In order to protect hypoxia-isolated donor cells from oxidative stress during injection, we injected hypoxia-isolated donor cells i.v. into B6 Boy/J F1 mice (CD45.1+CD45.2+) that had been lethally irradiated (950 cGy) 24 hours prior to transplantation and placed within the hypoxic glove box prior to i.v. injection. To achieve this, we used a mouse respirator/injector inside the hypoxic glove box (23). Mice were placed into an ambient air–filled, airtight container and passed through the air-lock system of our glove box. The animal was then very rapidly placed into the respirator that pumped normal ambient air to the mouse so that only the tail was exposed to hypoxic conditions. The animal was allowed to acclimate for several minutes prior to injection. After injection, the airflow was turned off, and the mouse was removed from the chamber in reverse of the above procedure. Ambient air–acclimated cells were injected under ambient air conditions. Finally, in a separate injection, 1.5 × 105 competitor BM cells isolated under ambient air conditions from Boy/J mice (CD45.1+CD45.2) were injected into all recipient mice under ambient air conditions (see experimental outline in Figure 2A). At the indicated time points, the percentages of donor CD45.1CD45.2+ cells in PB and BM were determined by flow cytometry. The myeloid/lymphoid (CD11b+/CD3e+B220+) ratio in donor cells (CD45.1CD45.2+) in the BM of engrafted mice was determined by flow cytometry at the 6-month point. For limiting dilution analysis, BM engraftment at 6 months was performed as previously described (62), and CRU frequency was calculated using L-Calc software (STEMCELL Technologies) and plotted using extreme limiting dilution analysis (ELDA) software (http://bioinf.wehi.edu.au/software/elda/). For secondary transplants, 1.5 × 106 BM cells from the above primary recipient mice were i.v. injected into lethally irradiated B6 Boy/J F1 mice in ambient air. The percentages of CD45.1CD45.2+ in PB and BM were evaluated at the indicated time points (see Figure 2A for the experimental schema).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.