Multiscreen hts ip plate

For ELISPOT assays, MoDC/T cell or MoDC/PBMC co-cultures were performed in 96-well MultiScreen HTS IP plates (0.45 μm, clear, Merck Chemicals GmbH, Darmstadt, Germany), coated with anti-IFNγ capture antibody (BD Biosciences, Heidelberg, Germany) overnight at 4°C. Following blocking for two hours at room temperature with culture medium, cells were seeded and stimulated as described above. After 16 h incubation, cells were discarded and plates washed in sterile water and PBS / 0.05% Tween20 (Sigma-Aldrich Chemie GmbH, Munich, Germany). Biotinylated anti-human IFNγ detection antibody (BD Biosciences, Heidelberg, Germany) was added in PBS / 10% FCS and plates incubated for two hours. After washing in PBS / 0.05% Tween20 Alkaline Phosphatase (AP)-conjugated Streptavidin (BD Biosciences, Heidelberg, Germany) was added 1:1000 in PBS/10% FCS followed by incubation for one hour. Development of the plate was performed with the AP conjugate substrate kit (Bio-Rad Laboratories GmbH, München, Germany); the reaction was stopped with water and the plate dried overnight.
FluoroSpot assay was carried out using the MabTech Kit (IFNγ/IL-13, MabTech, Stockholm, Sweden) following the provided protocol.
Spots and enzymatic activity were quantified with an iSpot FluoroSpot Reader System (AID, Strassberg, Germany).

Peptide-specific release of IFN-ɤ was assessed as previously described (9 (link)). Briefly, 96 Well MultiScreenHTS IP plates (0.45 mm, Merck Millipore, Darmstadt, Germany) were coated over night at 4°C with murine anti-IFN-ɤ antibody (clone AN18, Mabtech, Nacka Strand, Sweden (10 μg/ml)). After a washing step with PBS, the membrane was blocked with IMDM (Gibco, Carlsbad, USA) supplemented with 10% FCS, 1% glutamine and 1% sodium pyruvate for at least 60 min at 37°C. Spleen single cell suspensions were prepared as described before. 5x10⁵ splenocytes were incubated in the absence or presence of OVA_257-264 or OVA_323-337 peptides (each 1 μM). After 20 h of incubation at 37° C and 5% CO₂ the plate was washed six times with PBS including 0.05% Tween and the produced IFN-ɤ was detected with a biotinylated anti-IFN-ɤ antibody (clone R4-6A2, Mabtech, Nacka Strand, Sweden (2μg/ml)). After an additional washing step as described above, the enzyme-substrate reaction was performed to visualize the occurred binding using the Vectastain ABC Kit (Vector Laboratories, Burlingame, USA) together with AEC (Sigma-Aldrich, Taufkirchen, Germany) as described by the manufacturer. The reaction was stopped with H₂O. After drying, spot forming units (SFU) were measured with the automated image analysis system AID iSpot ELISpot reader (AID Autoimmun Diagnostika, Straßberg, Germany).