96 well transwell plate

For transwell experiments, CTLs were pre-incubated in RPMI with 0.5% bovine serum albumin for 90 min, placed in the upper chamber of a 96-well transwell plate with a pore size of 5 μm (Corning, Tewksbury, MA, USA) and allowed to migrate towards the bottom chamber containing the indicated concentrations of FBS for three hours. FBS was used as a general chemoattractant as suggested by the transwell system manufacturer (Corning). The fraction of cells that had migrated to the bottom chamber was subsequently determined by FACS analysis. The specific migration is given as the fraction of migrated cells towards the indicated concentration of FBS subtracted the fraction of migrated cells towards medium without FBS.

IL-2, IL-17, IFN-g, TNF-a and IL-10 from T cells and B cells were quantitatively measured by multiplex Luminex assay following protocols provided by manufacturer with modifications (EMD Millipore, Etobicoke, Canada). A total of 2x10⁵ T cells and/or B cells were plated in each well of 96-well plate (Corning, Tewksbury, MA, USA). For B cell stimulation, heat-killed H. pylori were added to the B cells, which were plated at the bottom of a 96-well transwell plate (Corning, Tewksbury, MA). For T cell stimulation, the bottom part of the transwell plate was pre-incubated with anti-human CD3 (clone OKT3) overnight and washed, after which purified T cells were transferred into the plate. Human cytokine capture antibody beads were added to the upper chamber of the 96-well transwell plate. Twelve hours later, the beads were harvested, washed and read according to manufacturer’s protocol.

CD4⁺ T cells were purified from lungs and spleen of BeO-exposed and spleen of PBS-treated mice at day 21. Tconv cells were purified from naive spleen. Teffs (CD44^hiCD25^lo) and Tregs (CD25⁺) were sorted from purified CD4⁺ T cells on FACS ARIA II fusion (BD Biosciences). Tconv cells were stained with 1 μM carboxy fluorescein isothiocyanate (CFSE; Invitrogen) and cocultured with Teffs or Tregs in a 1:1 ratio in a 96-well anti-CD3–coated (1–2 μg/mL, clone 145-2C11, Thermo Fisher Scientific) plates for 5 days. Transwell assays were performed in a 96-well transwell plate (Corning). Loss of CFSE was examined by flow cytometry at day 5.

An in vitro migration assay was performed to address factors mediating the migration of splenic NK cells to the liver. To this end, hepatocytes and sera derived from either naive or αGalCerMPEG treated mice (described above) were placed in the lower chamber of a 96-well trans-well plate (Corning). Sorted splenic NK cells (NK1.1 (PK136, PE, eBioscience), CD4 (RM4-5, BV421, BioLegend), CD8 (53-6.7, BV421, BioLegend), CD11c (HL3, FITC, BD), and B220 (RA3-6B2, FITC, eBioscience) isolated from untreated mice were stained with carboxyfluorescein succinimidyl ester (CFSE) and placed in the upper chamber of the trans-well system. After 2 h incubation at 37 °C, cells were collected from the lower chamber and stained for NK cells (CD3⁻ (500A2, V500, BD Horizon), NKp46⁺ (29A1.4, eFluor660, eBioscience)). For the determination of absolute cell numbers, CountBright™ Absolute Counting Beads (Thermo Fisher Scientific) were added to the samples prior to the acquisition.

Secreted IL-10 from purified B cells was quantitatively measured by IL-10 Luminex (EMD Millipore, Etobicoke, Canada). 10⁵ purified B cells were plated into the lower chamber of a 96-well transwell plate (Corning, Tewksbury, MA). For stimulation, TLR agonists were added to the B cells. Human IL-10 capture beads were added to the upper chamber of the 96-well transwell plate. 12 h later, the beads were harvested, washed and read according to manufacturer’s protocol.

pCAF2 cells were transfected as required and cultured for 24 h for recovery. AGS cells were transfected with pCS2+ Gap43-GFP (membrane-GFP) and also incubated for 24 h. The 3D invasion assays were performed using GrowDex® hydrogel (UPM) as the matrix supporting 3D cell growth. For the experiment, 1 × 10⁵ pCAF2 cells were resuspended in 40 µL complete cell culture medium (DMEM + 5% FBS, minus phenol red), and then mixed with 90 µL of the 1.0% hydrogel stock to achieve a 0.75% w/v hydrogel solution. Then, 80 µL of this suspension was dispensed to a well of a 96-well transwell plate (Corning) using a wide-bore pipette. The insert was placed on top, and 5 × 10³ transfected AGS cells aliquoted in 100 µL of DMEM + 5% FBS minus phenol red on top of the 8-µm pore-size membrane. Media were exchanged once per day for 72 h, and on the third day, cells were imaged using a Leica DMI6000 light microscope with a 20× dry objective. One-millimeter stacks were obtained, with a frame at every 5 µm. Images were analyzed using Imaris, with green AGS cells converted to spots and the depth of their position in the hydrogel measured. Three stacks were obtained in each well, and the experiments were performed in triplicate.