Spleens collected from naive 8‐week‐old KaLwRij mice were homogenised through a 70‐μm filter and subjected to hypotonic red blood cell (RBC) lysis. Splenocytes were treated with hMPO (2 μg/mL) in 20% FCS IMDM for 20 min then cultured at 2 × 105 cells/well in multiscreen‐IP HTS plates (Millipore) coated with mouse monoclonal anti‐IFN‐γ (Cat# R4‐6A2, MabTech, Sweden) with 1 × 105 5TGM1 cells for 36 h. Detection of IFN‐γ was achieved through the use of mouse monoclonal anti‐IFN‐γ (Cat#AN‐18, MabTech), streptavidin‐alkaline phosphatase and SigmaFast™ (Sigma Aldrich). Spot quantification was completed using an ELISpot reader (MabTech).
Expanded human Vγ9Vδ2 T‐cells were provided by Prof Andreas Evdokiou (Basil Hetzel Institute, The University of Adelaide).
30 (link) T‐cells were then treated for 2 h with hMPO in 10% heat‐inactivated FCS DMEM prior to being cultured at 1 × 104 cells/well in multiscreen‐IP HTS plates coated with human monoclonal anti‐IFN‐γ (Cat#1‐D1K, MabTech) with either RPMI‐8226 cells or MDA‐MB‐231‐TXSA cells at 1 × 103 cells/well for 16 h. Detection of IFN‐γ was achieved through the use of human monoclonal anti‐IFN‐γ (Cat#7‐B6‐1, MabTech) and developed as outlined above.
Expanded human Vγ9Vδ2 T‐cells were provided by Prof Andreas Evdokiou (Basil Hetzel Institute, The University of Adelaide).
30 (link) T‐cells were then treated for 2 h with hMPO in 10% heat‐inactivated FCS DMEM prior to being cultured at 1 × 104 cells/well in multiscreen‐IP HTS plates coated with human monoclonal anti‐IFN‐γ (Cat#1‐D1K, MabTech) with either RPMI‐8226 cells or MDA‐MB‐231‐TXSA cells at 1 × 103 cells/well for 16 h. Detection of IFN‐γ was achieved through the use of human monoclonal anti‐IFN‐γ (Cat#7‐B6‐1, MabTech) and developed as outlined above.