Multiscreen 96 well filtration plate

NP-specific ASCs in spleen or ASCs in the iPC differentiation system were detected using an ELISpot assay on a MultiScreen 96-well filtration plate (Millipore, MSIPS4W10) coated with 10 μg/ml NP₂₉-BSA or anti-mouse kappa-UNLB and anti-mouse lambda-UNLB (2.5 µg/ml). Serially diluted cells were added to individual wells in triplicate and incubated for 6 h at 37 °C with 5% CO₂. Anti-NP IgG1, Anti-IgG1 or Anti-IgE spots were revealed by biotin-conjugated anti-mouse IgG1 or IgE Ab (Southern Biotechnology, 1070-08) in conjunction with Av-HRP and AEC substrate (Vector Laboratories, A-2004& SK-4200).
For ELISA, flat-bottom 96-well plates (NUNC, 439454) were coated with NP-BSA (10 µg/ml) or anti-mouse kappa-UNLB and anti-mouse lambda-UNLB (2.5 µg/ml) in PBS. Nonspecific binding was blocked with 0.5% BSA in PBS. Serum samples were serially diluted in 0.5% BSA in PBS and were incubated in blocked plates at room temperature for 2 h. Plates were incubated with biotin-conjugated anti-Ig (Southern Biotech) for 2 h and with streptavidin–alkaline phosphatase for 1 h (Roche, 1089161), and then incubated with alkaline phosphatase substrate solution containing 4-nitro-phenyl phosphate (Sigma-Aldrich, N2765) for color development, followed by quantification on Molecular Devices (VERSA max).

The ELISpot-technique was applied for sensitive detection of IFNγ-secreting NK cells. First, a Multiscreen 96-well filtration plate (Merck Millipore) was activated with 35% ethanol and coated with anti-IFNγ capture antibody (200 µg/ml, clone 1-D1K, mIgG1, k, Mabtech). After incubation at 4 °C overnight, the plates were blocked with 200 µl of supplemented complete RPMI-1640-media for 2 h at 37 °C. After the washing step isolated NK cells were coincubated with IGROV1 cells in 1:1 ratio and either with or without ascites samples (ratio 1:4). For ADCC conditions, Cetuximab 1 µg/ml (Erbitux, 5 mg/mL, Merck (Serono) was added. After incubation for 24 h at 37 °C and 5% CO₂ plates were washed in the ELISA-washer (PBS / 0,05% Tween-20.) Biotinylated anti-IFNγ detection antibody (200 µg/ml, clone 7-B6-1, mIgG1, k, Mabtech) was added in 2 µg/ml PBS and 1% BSA. The plates were incubated for 2 h at 37 °C, washed and incubated with 50 µl ExtraAvidin alkaline phosphatase (1:1000 diluted in PBS / 1% BSA, Sigma-Aldrich) at room temperature for 2 h. After the washing steps, 75 µl of the ELISpot substrate BCIP/NBT (Roche) was added and incubated for 5–10 min. Developed cytokine spots were measured using AID Classic ELISpot Reader and the results were analyzed with AID ELISpot 7.0 software.

The ELISpot-technique was applied for sensitive detection of IFNγ-secreting NK cells. First, Multiscreen 96-well filtration plate (Merck Millipore) was activated with 35% ethanol and coated with anti-IFNγ-capture antibody (200µg/ml, clone 1-D1K, mIgG1, k, Mabtech). After incubation at 4°C overnight the plates were blocked with 200µl of supplemented complete RPMI-1640-media for 2h at 37°C. After washing step isolated NK cells were seeded in triplicates and treated with different nanoparticle types. NK cells treated with PMA (50ng/ml, Sigma-Aldrich) and Ionomycin (1µg/ml, Sigma-Aldrich) were included for positive control. Untreated NK cells were used as negative control. After incubation with 12,5 µl of calcium phosphate nanoparticles for 24 h at 37°C and 5% CO2 plates were washed in the ELISA-Washer (PBS/0,05% Tween-20.) Biotinylated anti-IFNγ-detection antibody (200µg/ml, clone 7-B6-1, mIgG1, k, Mabtech) was added in 2µg/ml PBS and 1% BSA. The plates were incubated for 2 h at 37°C, washed and incubated with 50µl ExtraAvidin alkaline phosphatase (1:1000 diluted in PBS/1% BSA, Sigma-Aldrich) for 2h at the room temperature. After washing steps 75µl of the ELISpot substrate BCIP/NBT (Roche) was added and incubated for 5-10 minutes. Developed cytokine spots were measured using AID Classic ELISpot Reader and the results were analyzed with AID ELISpot 7.0 software.

Multi screen 96-well filtration plates (Millipore, Billerica, MA) were coated with 4-hydroxy-3-nitrophenyl acetyl conjugated to bovine serum albumin (NP19-BSA, Biosearch Technologies, Petaluma, CA) at a concentration of 10 μg/ml overnight at 4°C. Splenocyte suspensions from immunized mice were initially plated at 1 × 10⁶ cells per well and then diluted serially (1:2) in RPMI1640 medium containing 10% FBS and incubated for six hours at 37°C. Bound antibodies were detected using biotinylated anti-mouse IgM and IgG Abs (Jackson Immunoresearch Laboratories, West, Grove, PA) followed by streptavidin conjugated to alkaline phosphatase (Vector Laboratories, Burlingame, CA). The plates were developed using the Vector Blue Alkaline–Phosphatase Substrate kit III (Vector Laboratories, Burlingame, CA). ELISpots were counted using a computerized imaging video system (Cellular Technology, Shaker Heights, OH).

Capture antibodies mouse IFN-γ and IL-4 cytokines (3 µg/mL in coating buffer, R&D Systems, USA) were coated on the Multiscreen 96-well filtration plates (Millipore, Billerica, MA, USA), respectively. Spleen cells (n = 3/group) were harvested at 2 weeks post prime-boost immunization. Freshly isolated splenocytes (1 × 10⁶ cells) were cultured on the plate with 100 µL of RPMI 1640 media with 10% FBS, and then stimulated with or without 10 μg/mL of HA-specific peptide (PKGRGLFGAIAGFIENGWEGL) for 36 h in a humidified 37 °C CO₂ incubator. After incubation, the plates were washed with sterile PBS and further incubated with biotinylated anti-mouse IFN-γ and IL-4 antibodies (1:5000 diluted). After three additional washes, alkaline phosphatase (AP) conjugated streptavidin (1: 1000) was added to each well and incubated at room temperature for 3 h. The plates were washed and developed with BCIP/NBT Chromogen (R&D Systems, USA). The number of IFN-γ and IL-4 secreting cells was counted using an ImmunoSpot ELISpot plate reader (Cellular Technology Ltd, Shaker Heights, OH, USA).