Trypan blue

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (Ficoll® Paque Plus, GE Healthcare, UK) density gradient centrifugation and were washed twice with DPBS (DPBS, Thermo Fisher Scientific, Carlsbad, California). Trypan Blue (STEMCELL Technologies, 0.4% (m/v), Cologne, Germany) staining was used for the discrimination between viable and non-viable cells and cell numbers were determined via Neubauer chamber.

Cell density was determined using Trypan Blue (Stemcell Technologies, Vancouver, BC, Canada) and a hemocytometer. Shh-Light II cells were seeded in a 96-well plate with 10,000 cells per well, and grown to complete confluence in the media described above. When cells were confluent, the media was replaced with DMEM supplemented with 0.5% bovine calf serum, and treated with 0.1 ng of N-terminal mouse recombinant Shh (R&D Systems, Minneapolis, MN, USA) dissolved in DMEM, and select alkaloid treatment. In each experiment, the controls and treatment wells contained all vehicles, with a final ethanol concentration of 0.05%. Gli activity in the Shh-Light II cell line was assayed 48 h after treatment with Shh protein and select compounds using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). The Gli-activity was measured by luminescence emitted from cells using a Synergy H1m Microplate reader (BioTek, Winooski, VT, USA). The Gli-activity determined in the biological assay is presented as a relative response ratio (RRR) as described in the Dual-Luciferase Reporter Assay System manual. Each experiment was performed three times.

Human PBMCs were freshly isolated from whole donor blood in K2EDTA anticoagulant Vacutainers (BD) using Lymphoprep density gradient medium (Stemcell) and SepMate tubes (Stemcell). PBMCs were enumerated in a haemocytometer with trypan blue 0.4% (Sigma) and the CD14+ CD16- monocytes were then magnetically separated using EasySep Human Monocyte Isolation Kit and EasySep Magnet following manufacturer’s instructions (Stemcell). Freshly isolated monocytes were then enumerated in a haemocytometer with 0.4% trypan blue and if viability was ≥90%, differentiation culture was established to differentiate the monocytes into dendritic cells using ImmunoCult Dendritic Cell Culture Kit following instructions of the manufacturer (Stemcell). According to the protocol (Stemcell), immature DCs used in the intracellular cytokine staining (ICS) co-culture did not receive maturation supplement on day 5 of culture and mature DCs used in the proliferation and memory co-culture received maturation supplement on day 5 of culture. After 7 days culture, immature DCs were used for the ICS co-culture and mature DCs were used for the proliferation co-culture.

For the co-culture of DCs and B cells, differentiated immature DC cells (iDCs) and B cells were harvested respectively at 6 d after culture respectively, and counted by staining with 0.4% Trypan blue (STEMCELL, Canada). About 1.0×10⁴ iDCs and 1.0×10⁵ B cells were seeded in the same wells of 96-well culture plates, and RPMI-1640 complete medium with 20 ng/ml rpGM-CSF, 25 μg/ml of rpIL-4, 50 μg/ml IFN-γ and 50 μg/ml CD40L were added. After co-cultured for 48 h, cells were under different treatment. In group 1, cells were infected with M.hp for 2 h. In group 2, cells were treated with rCTSL protein for 1 h followed by infection with M.hp for 2 h. In group 3, cells were transfected with eukaryotic plasmid CTSL-GFP for 48 h followed by infection with M.hp for 2 h. In group 4, cells were treated with E64 for 1 h followed by infection with M.hp for 2 h. In group 5, cells were transfected with px458-cas9-CTSL vector for 48 h followed by infection with M.hp for 2 h. At the same time, B cells and DCs were cultured separately as controls. The cell supernatants were collected for the detections of various cytokines and SIgA, and the pellets were collected for the detections of CTSL and Ii chain.

Sorted LSK were cultured in 96-well plates containing 100 µL StemSpan supplemented with 10% FBS and 1% penicillin/streptomycin, 1% L-glutamine, 100 µM β-mercaptoethanol (Sigma), 10 ng/mL TPO, and 50 ng/mL SCF (Peprotech Inc), with or without MMC. Cells were seeded at a density of 100,000/mL in triplicate and maintained at less than 1 million/mL throughout culture by subculture into fresh media. Viable cells were counted using a Hemacytometer (Hausser Scientific) using Trypan Blue (STEMCELL technologies).
Splenic B cells were cultured in 6-well plates containing 2.5 mL RPMI (Invitrogen) supplemented with 10% FBS, 1% penicillin/streptomycin, 1% nonessential amino acids (Life Technologies), 1% sodium pyruvate (Life Technologies), 50 µM β-mercaptoethanol, 5 ng/mL murine IL-4 (Life Technologies), 0.5 µg/mL RP105 (BD Pharmingen, 552128, 1:100), and 25 µg/mL LPS (Sigma).