Ultra low attachment flask

We obtained rat CSCs from the hearts of WKY rats as described in previously published papers^{12 (link),26 (link),27 (link)}. Briefly, hearts were minced into fragments less than 2 mm³, followed by digestion with collagenase and then seeded onto fibronectin-coated plates to create explant-derived cells. In about 14 days, explant-derived cells were harvested and seeded in ultralow attachment flasks (Corning Life Sciences, Durham, NC) and grown in suspended culture for cardiosphere formation. CSCs were obtained by replating cardiospheres on fibronectin-coated plates.

Monocytes were isolated from enriched leukocyte fractions of human peripheral blood purchased from the New York Blood Center using sequential Ficoll and Percoll density gradient centrifugations, as we have previously described [21 (link)]. Monocytes were cultured at 37°C and 5% CO₂ in ultra low attachment flasks (Corning) for five days at a density of 0.4×10⁶ cells/cm² and 1.0×10⁶ cells/ml of complete media (RPMI media supplemented with 10% heat-inactivated human serum, 1% penicillin-streptomycin, and 20ng/ml macrophage colony stimulating factor (MCSF)). Macrophages were polarized over the next 1–6 days by culturing at 1.0×10⁶ cells/ml in complete media with 100ng/ml IFN-gamma (Peprotech, Rocky Hill, NJ) and 100ng/ml lipopolysaccharide (LPS, Sigma Aldrich) for M1 or 40ng/ml IL4 and 20ng/ml IL13 (Peprotech, Rocky Hill, NJ) for M2, with a media change at day 3. At the media change, the media of another group of M1 macrophages was switched to M2-polarizing media and the media of a group of M2 macrophages was switched to M1-polarizing stimuli, in order to characterize the ability of macrophages to switch phenotypes. Unactivated macrophages were also cultured over the same time periods (M0), resulting in three groups through day 3 (M0, M1, M2) and five groups between days 4 and 6 (M0, M1, M2, M1→M2, M2→M1) (Fig. 1a).