Alexa fluor 488 affinipure f ab 2 fragment goat anti rabbit igg h l

Because ASC-speck formation is regarded as a simple upstream readout for inflammasome activation, the ratio of ASC-speck formation was evaluated as a criterion supporting inflammasome activation^{24 (link)}. Human PBMCs were incubated with 0.1 ng/mL LPS together with KN3014, KN8311, MCC950, or vehicle (DMSO) for 8 h. LPS-induced pro-IL-1β and cleavage of IL-1β were evaluated by western blotting using a rabbit anti-IL-1β (D3U3E) mAb #12703 (Cell Signaling Technology, Danvers, MA, USA) and a rabbit anti-cleaved-IL-1β (Asp116) (D3A3Z) mAb #83186 (Cell Signaling Technology), respectively. IL-1β concentrations in the supernatant of each well were measured in an ELISA. The cells in each well were fixed on glass slides and incubated with anti-ASC mouse mAb^{31 (link)}, followed by Alexa Fluor 488 AffiniPure F(ab′)₂ fragment goat anti-mouse IgG (H + L) (Jackson ImmunoResearch, West Grove, PA, USA) and monitoring by immunofluorescence microscopy to evaluate ASC-speck formation. Alternatively, the cells were incubated with a rabbit anti-NF-κB p65 (D14E12) XP mAb #8242 (Cell Signaling Technology), followed by Alexa Fluor 488 AffiniPure F(ab′)₂ fragment goat anti-rabbit IgG (H + L) (Jackson Immuno Research), and then monitoring by immunofluorescence microscopy to evaluate nuclear translocation of NF-κB.

Starved HL cells were seeded over isotype control or PD-L1 antibody-coated plates as described above. Forty-eight hours later, cells were in 4% formaldehyde for 10 min at 37 °C and then permeabilized in ice-cold methanol for 30 min on ice. Cells were then washed and incubated with rabbit mAb to phospho-p38 MAPK (Thr180/Tyr182) (Clone D3F9, Cell Signaling), rabbit mAB to phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E, Cell Signaling) or Isotype control IgG for 1 h at room temperature, followed with incubation with secondary Alexa Fluor^® 488 AffiniPure F(ab’)₂ fragment goat anti-rabbit IgG (H + L) (Jackson Immunoresearch Laboratories) for 30 min at room temperature. The intracellular levels of phospho-p38 and p-ERK MAPKs were analyzed using FACSCANTO II.