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AID ELISPOT plate reader software

Manufactured by Autoimmun Diagnostika

The AID ELISPOT plate reader software is a tool used to analyze and quantify the results of ELISPOT assays. It provides automated detection and evaluation of the spots generated in ELISPOT experiments, which are indicative of the presence and frequency of antigen-specific immune cells.

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4 protocols using AID ELISPOT plate reader software

1

Quantifying IFN-γ-Producing Cells in Peptide-Stimulated PBMCs

Enzyme‐linked immunospot assay was performed using a ELISpot kit (Mabtech) according to the manufacturer's instructions as described previously.29 Peptide‐stimulated human PBMCs (2 × 105) or mouse lymphocytes (5 × 103) were cultured in the presence of peptide (3 μg/ml) in a MAHAS4510 plate (Millipore) for 24 h, and the number of IFN‐γ‐producing cells in the culture was measured using an ELISpot kit (Mabtech) according to the manufacturer's instructions. BCIP/NBT plus substrate (Mabtech) was used for detection. Plates were scanned using an automated ELISpot plate reader (Autoimmun Diagnostika GmbH). Spots were counted and analyzed using AID ELISPOT plate reader software (Autoimmun Diagnostika GmbH). For mouse lymphocytes, splenocytes (3 × 105) were used as APC.
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PBMCs (1.5–2 × 106) derived from healthy donors were stimulated with 1 μg/ml of the Spike448-456 (short peptide binding to HLA-A24), the Spike448-456, or the Spike489-513 peptides in the presence of IL-2 (20 IU/ml) in 24-well plates, as described previously [16 (link)]. Seven days after peptide stimulation, the PBMCs were washed with PBS twice and addressed their specificity to the Spike peptides by an enzyme-linked immune absorbent spot (ELISpot) assay. The washed PBMCs (4 × 104) were cultured on a MAHAS4510 plate (Millipore) in the presence or absence of Spike peptides (1 μg/ml) for 24 h, and the number of IFN-γ-producing cells in the culture was measured using an ELISpot kit (Mabtech) according to the manufacturer's instructions. Plates were scanned with an automated ELISpot plate reader (Autoimmun Diagnostika GmbH). Spots were counted and analyzed using AID ELISPOT plate reader software (Autoimmun Diagnostika GmbH).
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3

Isolation and Functional Assay of Tumor-Infiltrating Monocytes/Macrophages

For purification of CD11b+Ly6C+ monocytes/macrophages from tumor tissues, single-cell suspensions from tumor tissues were prepared after 24 h of intratumoral treatment with control, anti-CD47 mAb, cGAMP, or a combination. CD45+ cells were isolated from the single-cell suspensions using the MACS isolation system (Miltenyi Biotech) according to the manufacturer’s protocol, and then CD45+CD11b+Ly6C+Ly6G monocytes/macrophages were sorted using FACSAria II (BD Biosciences). The sorted monocytes/macrophages were cocultured with OT-I cells in a MAHAS4510 plate (Millipore) for 24 h. IFN-γ–producing T cell numbers were visualized by anti-mouse IFN-γ enzyme-linked immunospot assay (ELISpot, catalog no. 3321-2A; Mabtech) according to the manufacturer’s protocol. BCIP/NBT plus substrate (Mabtech) was used for detection. Plates were scanned with an automated ELISpot plate reader (Autoimmun Diagnostika). Spots were counted and analyzed using the AID ELISpot plate reader software (Autoimmun Diagnostika).
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For purification of CD4+ T or CD8+ T cells, the spleens were harvested from mice bearing 4T1 tumors after 24 h of intratumoral treatment with control, S100, and S100 plus αTim-3. Single-cell suspensions were prepared using magnetic beads according to the manufacturer's protocol. The sorted CD4+ T or CD8+ T cells were cocultured with BMDCs and tumor cell debris in a 96-well plate for 24 h. IFN-γ-producing T cell numbers were visualized using anti-mouse IFN-γ ELISpot assay (Dakewe) according to the manufacturer's protocol. The spots were counted and analyzed using the AID ELISpot plate reader software (Autoimmun Diagnostika).
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