Dylight anti rabbit igg 680

BMDMs were plated at 1.56× 10⁵ cells per cm² on Costar Clear Not Treated 6-well plates with RPMI containing 10% L-cell supernatant/10% FBS. After 24 h, cells were challenged with L. pneumophila at MOI of 15, followed by contacting the BMDMs in the centrifuge at 400 RCF for 10 min. At the indicated time points, the medium was removed and adherent cells were lifted with ice-cold PBS and pelleted at 1,000 RCF for 5 min in the centrifuge. Cells were sorted and lysed with 1 × SDS Laemmli sample buffer (0.125 M Tris-Cl pH 6.8, 4% SDS, 20% glycerol, 10% beta-mercaptoethanol, 0.01% bromophenol blue). Cell lysates were boiled for 5 min and fractioned on SDS-polyacrylamide gels (Bio-Rad) and then electroblotted on nitrocellulose membranes. Blots were blocked with 5% BSA in Tris-buffered saline-tween (TBST: 0.05 M Tris-HCl pH 8.0, 0.138 M NaCl, 0.0027 M KCl, 0.05% Tween 20). For immune detection, cells were probed with a primary antibody (1:1,000 or manufacturer’s recommendation) in 5% BSA/TBST overnight at 4°C. After being washed with TBST, secondary antibodies Dylight anti-rabbit IgG 680, Dylight anti-mouse IgG 680, Dylight anti-rabbit IgG 800, or Dylight anti-mouse IgG 800 (Cell Signaling 1:20,000) were incubated in 4% milk/TBST for 45 min at room temperature. The membranes were scanned by Odyssey Imaging System and the Image Studio software (LI-COR Biosciences).