Lipopolysaccharide lps solution

CPT-isolated PBMCs from mutation carriers and sex-matched controls were thawed and plated 2.5 x 10⁶/ml in RPMI 1640 medium. The cells were allowed to rest for a minimum of 2 hours and thereafter the cells were washed once in PBS with 2% FBS, centrifuged at 550 x g for 2 minutes and stained with LIVE/DEAD™ Fixable Violet Dead Cell Stain Kit (Invitrogen; L34955). The cells were left unstimulated or stimulated with PMA/Cell Stimulation Cocktail (Invitrogen; 00-4975-93) or lipopolysaccharide (LPS) solution (Invitrogen; 00-4976-93) for 15 minutes, immediately fixed 10 minutes in 4% formaldehyde (Thermo Fisher Scientific; 28908) in PBS at RT and washed twice as above. Next, the cells were permeabilized with ice cold Perm Buffer III (BD Biosciences; 558050) for 30 minutes on ice, washed and finally stained for 35 minutes at RT: anti-human CD3 FITC (Thermo Fisher Scientific; 11-0036-42), anti-human CD4 PE Cy7 (BD Biosciences;557852) and anti-human NF-kB p65 Phosphorylation pS529 Alexa Fluor 647 (BD Biosciences; 558422). The data was measured with BD LSRFortessa™ using BD FACSDiva software and analyzed with FlowJo ™ 10 software (BD Biosciences).

Cell lines were treated with the following where relevant prior to downstream assays: 10 µg mL⁻¹ TGF‐β Receptor 1 small molecule inhibitor (LY 364 947, Tocris), 5 ng mL⁻¹ recombinant human TGF‐β1 (240‐B‐002, R&D Systems), 10 ng mL⁻¹ Lipopolysaccharide (LPS) solution (00‐4976‐93, Invitrogen), 10 ng mL⁻¹ recombinant human TNF‐α (PHC3015, Gibco), 10 ng mL⁻¹ recombinant human IL‐6 (PHC0064, Gibco), 10 ng mL⁻¹ recombinant human IL‐10 (PHC0104, Gibco), 10 ng mL⁻¹ recombinant human VEGF‐A (PHC9394, Gibco), 1 ng mL⁻¹ TNF‐α Receptor small molecule inhibitor (CAS 1049741‐03‐8, Calbiochem, Sigma–Aldrich).