Anti il17a alexafluor 647

Cryopreserved cells were thawed, washed, and permeabilized with Perm/Wash solution (BD Biosciences). Cells were then incubated at 4°C for 1 h with fluorescence-conjugated antibodies directed against surface antigens and intracellular cytokines. For detection of the transcription factor FOXP3, a nuclear permeabilization buffer (eBioscience, San Diego, CA, USA) was used in place of the Perm/Wash solution as per manufacturer’s protocol. The following fluorescence-conjugated antibodies were used: anti-CD3 PacBlue, anti-γδ TCR PE, anti-CD27 PECy7, anti-CD25 APC, anti-IFNγ Alexafluor 700, anti-IL17A Alexafluor 647, anti-Ki67FITC (BD biosciences, San Jose, CA, USA); anti-CD45RA QDot655, anti-CD8 QDot605, anti-CD4 QDot605 (Invitrogen, Eugene, OR, USA); and anti-CD39 FITC, anti-IL22 PerCP-Efluor710, and anti-FOXP3 PE (eBioscience, SanDiego, CA, USA).
The entire sample was acquired on a BD LSRFortessa Flow Cytometer using FACSDiva software (BD Biosciences, San Jose, CA, USA). Compensation was performed using compensation beads.

Cryopreserved cells were thawed, washed, and permeabilized with Perm/wash solution (BD Biosciences). Cells were then incubated at 4 °C for 1 hour with fluorescence-conjugated antibodies directed against surface antigens and intracellular cytokines. For detection of the transcription factor FOXP3, a nuclear permeabilisation buffer (eBioscience) was used in place of the Perm/wash solution as per manufacturer’s protocol. The following fluorescence-conjugated antibodies were used: anti-CD3 PacBlue, anti-GDTCR PE, anti-CD27 PECy7, anti-CD25 APC, anti-IFNγ Alexafluor 700, anti-IL17A Alexafluor 647, anti-Ki67FITC (all BD biosciences, San Jose, CA); anti-CD45RA QDot655, anti-CD8 QDot605, anti-CD4 QDot605 (all Invitrogen, Eugene, Or); anti-CD39 FITC, anti-IL22 PerCP-Efluor710, anti-FOXP3 PE (all eBioscience, SanDiego, CA).
The entire sample was acquired on a BD LSR Fortessa Flow Cytometer (model 649225B7, power 690 W, manufactured Sept 2010) configured with 4 lasers and 22 detectors using FACSDiva software (BD Bioscience, San Jose, CA, USA). Compensation for overlap of fluorescence detection was performed using compensation beads. Multiparameter panel development included evaluation of appropriate staining controls of antibody and fluorochrome interactions and of spectral overlap using control blood samples.