Cy5 goat anti rat igg h l

Cells were lifted from flasks by incubating in accutase (Innovative Cell Technologies, #AT104) for 5 minutes at 37°C followed by gentle agitation and brief centrifugation. Cells were re-suspended in primary antibody and incubated for 1 hour on ice. Cells were washed, centrifuged, and if applicable, were incubated in secondary antibody for 1 hour on ice. Following centrifugation, cells were re-suspended in DPBS supplemented with 1% heat-inactivated, sterile-filtered fetal bovine serum and analyzed using a FACSCalibur flow cytometer (BD Biosciences). All antibodies were diluted in DPBS supplemented with 2 mM CaCl₂ and 1.5 mM MgCl₂ and included the following: C3aR mouse mAb, clone 14D4 (Hycult, #HM1123, 1:50 dilution), purified monoclonal rat IgG2a, clone 54447.11 (R&D Biosystems, #MAB006, 1:100 dilution), Cy5 goat anti-rat IgG H+L (Jackson ImmunoResearch, #112-175-143, 1:200 dilution), FITC goat IgG to mouse Complement C3 (MP Biomedicals, #55500, 1:50 dilution), and FITC whole molecule goat IgG (Jackson ImmunoResearch, #005-090-003, 1:100 dilution). Gating strategy was based on cell viability, which was determined with an Annexin V – Propidium Idodide kit (BD Pharmingen, #556547). Data was analyzed with FlowJo software (FlowJo, LLC).

For fluorescent staining, the following secondary antibodies were used: Alexa Fluor 546 goat anti-Mouse IgG (H+L) (#A11030; Invitrogen, Carlsbad, CA; used at 1:500) and Cy5 goat anti-Rat IgG (H+L) (112-175-143; Jackson Immunoresearch, West Grove, PA; used at 1:400).