Anti interferon ifn γ pe cy7

At the beginning and conclusion of the study, peripheral blood samples were drawn into serum separator tubes (SST). LDL-C levels were quantified using the Cobas 6000 analyzer (Roche Diagnostics, Basel, Switzerland) through enzymatic colorimetric techniques.
For the analysis of PBMCs, we followed procedures based on a previously published article by our group (32 (link)). The study utilized several monoclonal antibodies, including anti-CD4-AF700, anti-CD8-PE, anti-CD8-APC, anti-CD28-APC, anti-CD57-FITC, anti-interferon (IFN)-γ-PE-Cy7, and anti-tumor necrosis factor (TNF)-α-APC, IL-17A-APC, Granzyme B-PE, perforin-APC, all sourced from eBioscience, San Diego, CA, USA. Following permeabilization, cells were stained for intracellular cytokines and cytotoxic molecules using anti-IFN-γ-PE-Cy7 and anti-TNF-α-APC. Multicolor flow cytometry was performed with the BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA). Data analysis was conducted using FlowJo V10 software (FlowJo, LLC, Ashland, OR, USA). Blood samples with a PBMC viability of 80% or more were used for FACS analysis, with 77 patients meeting this criterion at both baseline and follow-up visits.

PBMCs were pre-incubated with an anti-mouse CD16/32 Fc blocker (BD Pharmingen, USA), followed by anti-FVD-APC-Cy7 (all supplied by eBioscience, San Diego, CA, USA) to exclude dead cells. After washing with FACS staining buffer, cells were treated with fluorochrome-conjugated monoclonal antibodies for 40 min at 4°C. The monoclonal antibodies used in this study were as follows: anti-CD3-PerCP-Cy5.5, anti-CD3-PE-Cy7, anti-CD4-AF700, anti-CD8-PE, anti-CD8-APC, anti-CD28-APC, anti-CD45RA-FITC, anti-CD45RO-PE-Cy7, anti-CD57-FITC, anti-TCR gamma/delta-FITC, fixable viability dye-APC-Cy7, anti-interferon (IFN)-γ-PE-Cy7, and anti-tumor necrosis factor (TNF)-α-APC (all supplied by eBioscience, San Diego, CA, USA). For intracellular staining, surface-stained cells were stimulated with phorbol-myristate acetate/ionomycin/brefeldin A/monensin for 5 h, and then fixed and permeabilized using a Fixation/Permeabilization Buffer kit (eBioscience, San Diego, CA, USA). The permeabilized cells were washed and resuspended in 1% formaldehyde and stained with anti-IFN-γ-PE-Cy7 and anti-TNF-α-APC. Multicolor flow cytometry was performed using a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA), and the data were analyzed by FlowJo V10 software (FlowJo, LLC, Ashland, OR, USA).