Serum-free CM was prepared by incubating serum-free medium (BIT; STEMCELL Technologies), 40 μg/ml low-density lipoproteins (Sigma), 100 U/ml penicillin, 100 μg/ml streptomycin, and 10−4 M β-mercaptoethanol for 3 days on irradiated (30 Gy) confluent pLKO.1 (pLKO.1-CM) and shCtgf (shCtgf-CM) stromal cells. Before use, CM was filtered through a 0.4 μm filter.
CD34− and CD34+ SLAM cells were sorted into round-bottomed 96-well plates preloaded with 100 μl of pLKO.1-CM and shCtgf-CM, supplemented with mSCF (100 ng/ml) and IL-11 (20 ng/ml), both from R&D Systems, and rCTGF (250 ng/ml; BioVendor) where indicated. Immediately after sorting, the plates were centrifuged for 5 min at 200 × g and microscopically inspected for the presence of single cells. Each well was inspected every 24 hr for clonal growth. After 5 days, each clone was harvested and studied for colony formation in growth factor-supplemented methylcellulose (M3434; STEMCELL Technologies), After 10 days, the number of colonies was counted and cells were harvested, and washed three times with HF2+, pelleted, and stained with B220-PECy7, CD3ε-PECy5.5, CD11b-APCCy7, GR1-PB, and TER119-PE (eBiosciences). Immunofluorescence staining was measured on a CyAn ADP Lx P8 (Coulter-Cytomation) and analyzed with FlowJo software (TreeStar).
CD34− and CD34+ SLAM cells were sorted into round-bottomed 96-well plates preloaded with 100 μl of pLKO.1-CM and shCtgf-CM, supplemented with mSCF (100 ng/ml) and IL-11 (20 ng/ml), both from R&D Systems, and rCTGF (250 ng/ml; BioVendor) where indicated. Immediately after sorting, the plates were centrifuged for 5 min at 200 × g and microscopically inspected for the presence of single cells. Each well was inspected every 24 hr for clonal growth. After 5 days, each clone was harvested and studied for colony formation in growth factor-supplemented methylcellulose (M3434; STEMCELL Technologies), After 10 days, the number of colonies was counted and cells were harvested, and washed three times with HF2+, pelleted, and stained with B220-PECy7, CD3ε-PECy5.5, CD11b-APCCy7, GR1-PB, and TER119-PE (eBiosciences). Immunofluorescence staining was measured on a CyAn ADP Lx P8 (Coulter-Cytomation) and analyzed with FlowJo software (TreeStar).