Anti cd8a pe

Mouse cell suspensions were stimulated with 50 μg/mL of TT or HEL peptide in the presence of BD GolgiPlug™ (BD Biosciences) for 2 h (37 °C). Cells were fixed and stained using the BD Cytofix/Cytoperm™ Kit (BD Biosciences). Surface and intracellular staining (ICS) were performed according to the manufacturer’s instructions, with anti-CD4-FITC, anti-CD3-APC-eFluor^®780, anti-CD19-PECy7, anti-TNF-α-PerCP-e710, anti-IL-2-APC and anti-IFNγ-EF450 from eBioscience and anti-CD8a-PE from BD Biosciences.

APC-anti-CD3e, PE-Cy7-anti-CD3e, FITC-anti-CD3e, PE-anti-CD8a, PerCp-anti-CD8a, Alex700-anti-CD19, FITC-anti-CD44, APC-anti-CD44, V450-anti-CD44, PE-anti-IFN-γ, PE-Cy7-anti-TNF-α were purchased from BD Biosciences (Australia); PE-Cy7-anti-CD25, FITC-anti-CD27, APC-anti-CD27, Alexa647-anti-CD107a, eFluo450-anti-CD122, PE-anti-T-bet and Alexa700-anti-Eomes and purified anti-mouse MHC I-SIINFEKL complex (clone 25D) were purchased from eBioscience (Jomar Bioscience, Kensington, SA., Australia); PerCp-Cy5.5-anti-CD62L, PE-Cy7-anti-CD69 and purified anti-CD11c were purchased from BioLegend, (Australian Biosearch, Karrinyup, WA., Australia); APC-H-2Kb-SIINFEKL pentamer was purchased from ProImmune (Oxford, UK).

The flow cytometry procedure for identification and quantification of circulating inflammatory and immune cells was based on our previous report [34 (link)]. Prior to sacrificing the animals, peripheral blood mononuclear cells (PBMCs) were obtained from the tail vein using a 27# needle. PBMCs (1.0 × 10⁶ cells) were triple-stained with FITC-anti-CD3 (BioLegend), PE-anti-CD8a (BD Bioscience, San Jose, CA, USA), and PE-Cy™5 anti-CD4 (BD Bioscience, San Jose, CA, USA). To identify CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs), PBMCs were triple-stained with Alexa Fluor® 488-anti-CD25 (BioLegend, San Diego, CA, USA), PE-anti-Foxp3 (BioLegend, San Diego, CA, USA), and PE-Cy™5 anti-CD4 (BD bioscience, San Jose, CA, USA) according to the manufacturer's protocol for the Foxp3 Fix/Perm buffer set. The numbers of CD3⁺CD4⁺ helper T cells, CD3⁺CD8⁺ cytotoxic T cells and CD4⁺CD25⁺Foxp3⁺ Tregs were analyzed using flow cytometry (FC500, Beckman Coulter, Brea, CA, USA).
Additionally, the numbers of inflammatory cells in circulation [i.e., CD11b/c, LyG6, vascular cell adhesion molecule (VCAM)-1], in ascites [macrophage migratory inhibitor factor (MIF), CD14, CD11b/c, LyG6, CD68/CD80, CD68/CD163], and in ABL (CD11b/c, MIF, Ly6G) were assessed using the flow cytometric method.

The procedure and protocol of flow cytometry for identification and quantification of circulating and splenic immune cells were based on our previous report [44 (link)]. Briefly, the peripheral blood mononuclear cells (PBMCs) and splenocytes were obtained from the tail vein using a 27# needle. PBMCs and splenocytes (1.0 × 10⁶ cells) were triple-stained with FITC-anti-CD3 (BioLegend), PE-anti-CD8a (BD Bioscience), and PE-Cy™5 anti-CD4 (BD bioscience). The numbers of CD3⁺CD4⁺ helper T cells and CD3⁺CD8⁺ cytotoxic T cells were analyzed using flow cytometry (FC500, Beckman Coulter).