3 amino 9 ethylcarbazole aec substrate

The levels of OVA-specific T cells were quantified by ELISpot using a mouse interferon (IFN)-γ kit (Mabtech Inc., Cincinnati, OH, USA) as described [25 (link)]. To obtain a sufficient number of lymphocytes for measurement of antigen-specific T cells, spleens were mechanically minced with the frosted ends of two glass slides in R10 media. The splenocyte cell suspension was passed through a 70 µm cell strainer and cell concentrations determined on a Cellometer (Nexcelom, Lawrence, MA, USA). The 4 × 10⁵ cells were stimulated in duplicate with peptides corresponding to the CD8⁺ T cell epitope OVA_257–264: SIINFEKL or the CD4⁺ T cell epitope OVA_266–277: TEWTSSNVMEER at a final concentration of 2 µg/mL. Both these epitopes were contained within the long peptide antigen used for immunization. Final volume per well was 0.2 mL. Cells were also incubated without any stimulants to measure background responses. Plates were incubated for ~20 h at 37 °C with 5% CO₂. Then, the plates were washed and developed according to the manufacturer’s instructions. 3-Amino-9-ethylcarbazole (AEC) substrate (Becton Dickinson, Franklin Lakes, NJ, USA) was used to visualize the spots. Spots were counted using an automated ELISpot plate reader.

IFN-γ ELISPOT assay kit (BD Biosciences, USA) was used as described by the manufacturer. Plates were coated with anti-IFN-γ mAb overnight at 4°C and then blocked with RPMI 1640 medium containing 10% fetal bovine serum (FBS) for 1 h at room temperature. Splenocytes (2.5 × 10⁵ cells/well) from immunized mice were isolated, plated, and cultured with 10 μg/mL PPD (Statens Serum Institute, Denmark) or 2 μg/mL recombinant Ag85A, 6 μg/mL recombinant ESAT-6 to provide stimulation at 37°C, 5% CO₂ for 20 h. After washing the plates with PBS-T20 (1 × PBS, pH 7.4, 0.05% Tween 20), biotinylated anti-IFN-γ was added for 2 h at room temperature. Streptavidin-HRP was added for 45 min, and the color was developed with 3-amino-9-ethylcarbazole (AEC) substrate (BD Biosciences). An immunospot analyzer (Cellular Technology, USA) was used to count the spots.

Splenocytes were analyzed for IFN-γ and IL-10 production in response to antigen-specific stimulation via ELISPOT kits (BD; Franklin Lakes, NJ) according to manufacturer’s instructions. Briefly, cells were plated in triplicate at 3 × 10⁵ cells/well with the following stimulatory conditions: 1) unstimulated; 2) recombinant human insulin (1 μg/ml) (Sigma-Aldrich Co. LLC; St. Louis, MO); 3) recombinant human GAD (1 μg/ml) (KRONUS Inc.; Star, ID); 4) recombinant CTB (1 μg/ml) (Sigma-Aldrich Co.); and 5) soluble anti-CD3 (5 μg/ml) + anti-CD28 (2.5 μg/ml) (eBioscience). Cells were incubated at 37 °C and 5% CO₂ for 65 hours. Cytokine spots were developed (6 minutes for IFN-γ and 15 minutes for IL-10) using 3-amino-9-ethylcarbazole (AEC) substrate (BD) according to manufacturer’s instructions. Spots were enumerated using a Bioreader^® 4000 Pro-X and the Bioreader^® software generation 8 (BIOSYS USA LLC; Miami, FL). Spots per well were summed, averaged across triplicates, and reported as a stimulation index relative to unstimulated cells.

Antibody-secreting cells (ASCs) in blood and bone marrow were analyzed by performing the enzyme-linked immunospot assay (ELISPOT) as described in detail elsewhere [21 (link)]. Briefly, 96-well MultiScreen_HTS HA filter plates (Millipore) were coated with 10 µg/mL of anti-monkey IgG (H&L) antibody (Rockland) to determine the total ASCs or with 10 µg/mL of polio virus–specific antigens (Sanofi Pasteur) to determine the antigen-specific ASCs. After the overnight coat at 4°C, the plates were washed with PBS/0.05% Tween 20 (PBS-T) followed by PBS and blocked for 2 hours with complete RPMI at 37°C. The lymphocytes were then plated with 3-fold serial dilutions and kept in a 5% CO₂ incubator at 37°C for 5 hours. The plates were then washed with PBS followed by PBS-T and incubated with biotin-conjugated anti-monkey IgG (Rockland) for 2 hours at room temperature. The plates were washed with PBS-T and incubated for 3 hours at room temperature with Horseradish Peroxidase Avidin D (Avidin D-HRP; Vector labs). The plates were then washed with PBS-T followed by PBS and developed with 3-amino-9-ethylcarbazole (AEC) substrate (BD Biosciences) according to the manufacturer’s protocol. Plates were then dried, and spots were imaged and counted using Immunospot Cellular Technology Limited (CTL) counter and Image Acquisition 4.5 software (Cellular Technology).

Purified splenocytes or BM cells were added at different dilutions to a 96-well 0.45-μm PVDF membrane (Millipore, cat#MSIPS4W10) previously coated overnight at 4 °C with 2 μg/mL NP₂₀BSA and blocked with complete RPMI cell culture media for 2 h at 37 °C. Plates with cells were incubated in a humid chamber 12 h at 37 °C, 5% CO₂, then washed 6× with PBS 0.01% Tween-20, followed by incubation with goat anti-mouse IgG1-HRP (A10551, Life Technologies, 1/2000) diluted in culture media for 2 h at RT. Plates were washed and AEC substrate (3′ amino-9-ethylcarbazole; BD Bioscience) was added to reveal the spots. Images were acquired in an Axiophot MZ12 microscope and scored spots were counted from the 2 × 10⁶ cells dilution.

Purified splenocytes or bone marrow (BM) cells were added at different dilutions to a 96-well 0.45 μm polyvinylidene difluoride membrane (cat. #MSIPS4W10; Millipore) previously coated overnight at 4°C with 2 μg/ml NP₂₀BSA and blocked with complete RPMI cell culture media for 2 h at 37°C. Plates with cells were incubated in a humid chamber 12 h at 37°C, 5% CO₂, then washed six times with PBS 0.01% Tween-20, followed by incubation with goat anti-mouse IgG1-HRP (A10551, 1/2,000; Life Technologies) diluted in culture media for 2 h at room temperature. Plates were washed and AEC substrate (3′ amino-9-ethylcarbazole; BD Bioscience) was added to reveal the spots. Images were acquired in an Axiophot MZ12 microscope and scored spots were counted from appropriate cell dilutions (2 × 10⁶ cells after primary immunization and 0.5 × 10⁶ cells for recall).