Negative isolation from primary culture
Trypsinized and washed RA and OA synovial cells from primary culture (107/ml) were incubated with 4 × 107/ml Dynabeads® M-450 CD14 (clone RMO52; Dynal) in PBS/2% FCS for 1 h at 4°C under bidirectional rotation. Nine milliliters of PBS/2% FCS were then added and the conjugated cells collected using the Dynal magnetic particle concentrator®. Magneto-bead-conjugated cells and unconjugated cells were collected and washed twice in PBS/2% FCS; cell composition and phenotype were analyzed by flow cytometry using the antibodies presented in Table 3. For the purpose of comparison with fourth-passage cells obtained by conventional isolation (ie repeated passaging; see later), negatively isolated RA-SFB and OA-SFB were then passaged four times by culture in complete DMEM/10% FCS (see earlier) with a 1:3 split of confluent cells in each passage.