Alexa fluor 488 conjugated antibody

PBMCs isolated by Ficoll-Paque gradient centrifugation from mutation carrier and sex-matched controls were thawed and plated 3 x 10⁶/ml in RPMI 1640 medium. The cells were allowed to rest for 2 hours and thereafter live and dead cells were stained with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (Invitrogen; L10119). The cells were washed twice in PBS (Sigma Aldrich; D1408) with 2% FBS and centrifuged at 550 x g for 2 minutes. The cells were stained for surface markers in Brilliant Stain Buffer (BD Biosciences; 563794) for 30 minutes at 4°C, washed, fixed and permeabilized with Transcription Factor Staining Buffer Set (Miltenyi Biotec, 130-122-981) for 30 minutes at 4°C. After permeabilization the cells were washed and stained for FOXP3 for 35 minutes at 4°C. The antibodies against anti-human CD4 BV510 (562970), anti-human CD127 PE Cy7 (560822), anti-human CD45RA APC (561210), anti-human CD25 BV421 (BD Biosciences; 564033), anti-human CCR7 PE (566741), anti-human CCR6 PE (551773) and anti-human CXCR3 PE (550633) were purchased from BD Biosciences. The Alexa Fluor 488 conjugated antibody against human FOXP3 was purchased from Biolegend (Biolegend; 320112) The data was collected with BD LSRFortessa™ using BD FACSDiva software and analyzed with FlowJo™ 10 (BD Biosciences).

Standard techniques for immunofluorescence were used to stain for pancytokeritin (mouse monoclonal anti-pancytokeratin FITC–conjugated antibody, Upstate Millipore Corp., USA), EpCAM (mouse monoclonal anti-CD326 Alexafluor® 488–conjugated antibody, Biolegend, USA), CA125 (mouse monoclonal anti-CA125 antibody, Abcam, USA, and Alexafluor^® 546 goat anti-mouse secondary antibody, Invitrogen, USA), MOC-31 (mouse monoclonal anti-MOC-31 antibody, Dako, Germany, and Alexaflour^® 596 goat anti-mouse secondary antibody, Invitrogen, USA), D2-40 (mouse monoclonal anti-D2-40 antibody, Dako, Germany, and goat anti-mouse Alexafluor^® 596 secondary antibody, Invitrogen, USA) and vimentin (rabbit monoclonal anti-vimentin antibody, clone EPR3776, Abcam, USA, and Alexafluor^® 488 goat anti-rabbit secondary antibody, Invitrogen, USA).