Cellometer k2 cell counter

As described previously, mice were euthanized and lungs perfused with 10 ml 1× Dulbecco's phosphate-buffered saline without calcium or magnesium (Gibco) (PBS) containing 50 U/ml heparin (Sigma) and placed into 2 ml complete Dulbecco’s modified Eagle’s medium (c-DMEM), DMEM (10-017-CV, Corning), 500 ml supplemented with filter-sterilized 5 ml HEPES buffer (1 M; Sigma), 10 ml MEM nonessential amino acid solution (100×; Sigma), 5 ml penicillin-streptomycin (pen/strep) (100×; Sigma), 660 μl 2-mercaptoethanol (50 mM; Sigma), and 45 ml heat-inactivated fetal bovine serum (FBS) (Atlas Biologicals). A single-cell suspension was obtained using enzymatic digestion (49 (link)). Residual erythrocytes were lysed using Gey’s solution (8 mM NH₄Cl, 5 mM KHCO₃ in water), passed through a 40-μm strainer, and suspended in c-DMEM. The total number of viable cells was determined with acridine orange and propidium iodide (AO/PI) staining and counted on a Cellometer K2 cell counter (Nexcelom Bioscience).

Mixed bone marrow chimeras were set up as previously described (32 (link)). Recipient mice were irradiated with 9 Gy in a MultiRad 350 (Faxitron), with 350 kV, 11.4 mA, a Thoraeus filter [0.75 mm Tin (Sn), 0.25 mm Copper (Cu), and 1.5 mm Aluminium (Al)], and with a beam-distance of 37 cm. Irradiated recipients were kept on antibiotic water (either 1 mg sulfadiazine together with 0.2 mg trimethoprim per mL drinking water, or 0.25 mg amoxicillin per mL drinking water) to avoid any opportunistic infections. On the following day, donor mice were anesthetized with 4% isoflurane and euthanized. Femora, fibulae/tibiae, ossa coxae and humeri were harvested, mechanically cleaned and rinsed in FACS buffer. The bone marrow (BM) cells were released from the harvested bones by crushing and the cell extract was then passed through a 70 µm cell strainer. The donor BM cells were then counted in a Cellometer K2 cell counter (Nexcelom). Cells were pelleted by centrifugation (200 g, 10 min, 4°C) and resuspended to 1*10⁸ cells/mL. Donor cells from three different mice were then mixed according to the proportions mentioned in the figure legend. The donor cell mixtures were used to reconstitute the recipient mice by retroorbital injection of 200 µL (containing a total of 20*10⁶ cells) into each recipient mouse. The reconstituted recipient mice were placed on antibiotic water the following 14 days.