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… Where indicated, cells were also treated with nigericin (5 μM, Invivogen, 10–45 min), CTB (20 μg ml−1, List Biological Laboratories, 6 h), silica (200 μg ml−1, Invivogen, 6 h), TcdB (10 μg ml−1, R&D Systems, 8 h) and ATP (5 mM, Millipore-Sigma, 25 min); cultured in K+-free medium (0.8 mM MgCl2, 1.5 mM CaCl2, 10 mM HEPES, 5 mM glucose and 140 mM NaCl, pH 7.2, 3 h)50; or transfected with flagellin (500 ng ml−1, Invivogen, 4 h), poly(dA:dT) (1 μg ml−1, Invivogen, 4 h) or FSL-1 (0.2 μg ml−1, Invivogen, 4 h) and ultrapure LPS (1 μg ml−1, 4 h) with Lipofectamine 2000 (Invitrogen) or as otherwise indicated.
… XCR1+ cDC1 cells were depleted by injecting Xcr1DTR mice with 500ng un-nicked DT (150, List Biologicals) intraperitoneally in 100µl PBS. DT was typically administered 2d before induction of MICB expression on tumour cells by doxycycline treatment and then every 48h until the study endpoint. …
Diphtheria toxin (DT) and puromycin treatments in vitro
Transfected HEK293 cells for human diphtheria toxin receptor (hDTR) mutagenesis were selected with 20 ng/ml DT (List Labs, #150) from day 3 after electro-transfections. Cell clones mutated in hDTR-sgRNA-1 or hDTR-sgRNA-2 targeted regions were cultured with DT-supplemented growth medium from day 1 after cell sorting (day 0 refers to the day of cell sorting). Transfected PFFs were selected with 300 ng/ml puromycin-containing (MPBIO, 219453925) medium for 48 h after electro-transfections of 12 h. Both DT and puromycin-supplemented growth medium were exchanged daily until negative control cells died completely.
EAE was induced by subcutaneous immunization of mice into the flanks with an emulsion of MOG35-55 peptide (150 μg) emulsified in complete Freund adjuvant supplemented with 4mg/ml of M. tuberculosis extract H37Ra (Difco). In addition, the animals received 200 ng of pertussis toxin (List Biological Laboratories) i.p. on days 0 and 2. …
Whey Sensitization/Challenge Protocol and Assessment of Anaphylaxis
The model of cow’s mike allergy-induced autism was established as described by de Theije’s et al, with modifications.37 Whey is a milk protein fraction composed of several milk allergens (β-lactoglobulin, α-lactalbumin, bovine lactoferrin, bovine serum albumin, and bovine immunoglobulins), some of which are major allergens, and used to induce our food allergy model.38 After 1 week acclimation, mice were sensitized intragastrically (i.g) with 20 mg whey (Hefei Bomei Biotechnology Co., LTD, China) in 0.5 mL of PBS plus 10 μg cholera toxin (CT, List Biological Laboratories, Campbell, CA, USA) as an adjuvant from week 1 through week 5 and oral 100 mg whey containing 10 μg CT from week 6 through week 7. Mice were then sensitized via intraperitoneal injection (i.p.) with 500 μg whey in 0.2 mL of PBS containing 2 mg Alum (ThermoFisher, USA) from week 8 through week 9. Naive mice received PBS alone. Sham mice received CT and alum alone. A schematic for the sensitization and challenge timeline was depicted in Figure 1. …