Complement C5a Quantification from Conditioned Media

BMMNCs from WT or Casp1-KO or NLRP3 KO mice injected with the mixture of interleukins or interleukins and DAMPs in the presence or absence of AMD3100 or G-CSF were isolated as described above. The residual RBCs were lysed using Lyse buffer (BD Biosciences, San Jose, CA, USA). Then, the samples were incubated in 0.5% BSA containing RPMI for 24 h at 37 °C in a 5% CO2 incubator. The culture supernatant was collected. A 100 µl of the conditioned medium samples were used in this experiment [24 (link), 28 (link)]. The experiments were performed in triplicates using complement C5a mouse ELISA kit (Abcam, Cambridge, MA, USA) as described in manufacturer’s protocol.