Elisa maxtm deluxe set mouse tnf α

Confluent monolayers of J774 macrophages in a 96-well plate were infected with AC or PC of At17-14 with 2 or 10 spores per macrophage, respectively, and incubated for 6 h at 37°C. Cell-free supernatants were harvested and the TNFα concentration was determined using the ELISA MAXTM Deluxe Set Mouse TNF-α (BioLegend, San Diego, USA). Analysis was carried out in triplicates using 25 μl of sample volume and a dilution of 1:10 according to the protocol of the manufacturer, followed by the addition of an avidin-HRP-labeled secondary antibody and TMB substrate solution. Absorbance reading was performed in a NanoQuant infinite M200 pro microplate reader (Tecan, Crailsheim, Germany) at 450 and 570 nm.

McLean, China was the source of Iron(III) acetylacetonate, 1,2-hexanediol, oleamine and benzyl ether. Niacin and BSA were bought from China’s Aladdin. IR780 dyes and oleic acid were from Sigma-Aldrich, USA. Local companies supplied other chemical reagents. Mouse breast cancer 4T1 cell line was obtained from the American Type Culture Collection. Trypsin EDTA solution (0.25%), fetal bovine serum and penicillin streptomycin were purchased from Gibco, USA. Calcein-AM/PI Staining Kit and Cell Counting Kit-8 were bought from Biyuntian Biotechnology Co., Ltd, China. Hoechst 33342 and Perls dye were purchased from Solarbio Technology Co., Ltd, China. ELISA MAXTM Deluxe Set Mouse TNF-α and ELISA MAXTM Deluxe Set Mouse IFN-γ were purchased from BioLegend, USA. Animal experiments were approved by the Laboratory Animal Management Committee of Sun Yat-sen University.

Cytokine levels in BALF and serum were assessed by ELISA using ELISA MAX^TM Deluxe Set Mouse IFN‐α1 (BioLegend), ELISA MAX^TM Deluxe Set Mouse TNF‐α (BioLegend), BD OptEIA^TM Mouse IFN‐γ ELISA Set (BD Bioscience), DuoSet Mouse IL‐6 (R&D Systems), Invitrogen Mouse IL‐10 Uncoated ELISA (Invitrogen), and Invitrogen Mouse IL‐22 Uncoated ELISA (Invitrogen). All assays were performed according to the manufacturer's instructions.