Anti tnf α alexa fluor 488

Splenocytes were isolated for analysis via IFN-γ ELISpot, surface and intracellular cytokine staining (ICS) and flow cytometry as previously described^{69 (link)–71 (link)}. Splenocytes were restimulated with 2 μg/mL of the appropriate antigenic peptide pool (comprised of individual peptides that were 20 amino acids in length with 10 amino acids of overlap) spanning the full-length LASV GPC sequence (Mimotopes). Reference sequences for peptide synthesis are as follows: NP_694870.1 (Josiah), AIT17836.1 (Pinneo), AAF86703.1 (803213), CAA36645.1 (GA391). Cells were restimulated for 18–20 h or 6 h (at 37 °C and 5% CO₂) for IFN-γ ELISpot and ICS, respectively. Surface staining and ICS were carried out using the following antibodies: LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (Thermo Fischer Scientific); anti-CD8a-PerCP/Cy5.5, anti-CD62L-PeCy7, anti-IFN-γ-eFluor 450, anti-TNF-α-Alexa Fluor 488, anti-IL-2-PE (eBioscience); anti-CD4-Brilliant Violet 650, anti-CD44-Alexa Fluor 700 (BioLegend); and anti-CD127-eFluor 660 (Invitrogen). Antigen-specific T-cell responses were quantified by subtracting the response (SFU for IFN-γ ELISpot and the percentage of cytokine-positive cells for ICS) measured without stimulation from that observed after restimulation.