Flow cytometric analysis of 2% paraformaldehyde fixed, 0.1% Tween-20 permeabilized Human peripheral blood mononuclear (PBMC) (treated with 10ng/ml IFN-gamma for 1h, then add 300ng/ml Brefeldin A (BFA) for another 3h) cells labelling IP10 with ab254374 at 1/50 dilution (1 ug) (Right) compared with a Rabbit monoclonal IgG  (ab172730) isotype control (Left). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Cells were stained with anti-CD14 conjugated to BV510. Then fixed with 2% PFA followed by intracellularly stained with rabbit IgG (Left) or ab254374 (Right).

IP10 was immunoprecipitated from 0.35 mg THP-1 (human monocytic leukemia monocyte) (treated with 200ng/ml IFN-r and 50ng/ml LPS for 24 hours) supernatant with ab254374 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254374 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: THP-1 treated with 200ng/ml IFN-r and 50ng/ml LPS for 24 hours, supernatant 10 ug

Lane 2: 254374 IP in THP-1 treated with 200ng/ml IFN-r and 50ng/ml LPS for 24 hours, supernatant

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254374 in THP-1 treated with 200ng/ml IFN-r and 50ng/ml LPS for 24 hours, supernatant

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 3 minutes.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized THP-1 (Human monocytic leukemia monocyte) (treated with 200ng/ml IFN gamma, 50ng/ml lipopolysaccharide (LPS) and protein transport inhibitor Cocktail for 24h) (Right) / Untreated control (Left)  labelling IP10 with ab254374 at 1/50 dilution. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.