Trypsine

All the manipulations were carried out by two independent operators with, respectively, 4 and 27 years of experience in cell cultures and experimental studies. Cell lines were detached (Day 0) with a mixture of 0.05% trypsine/0.5 mM EDTA (Sigma-Aldrich, Saint-Louis, MO, USA) after rinsing with Dulbecco’s Phosphate Buffered Saline (PBS). Cells were seeded onto a 96-well tissue culture-plate, 10,000 cells per well, and cultured for 24 h. At Day 1, the culture medium was changed to lower the FBS level to 0.5%. Drugs with the maximal concentration were deposited in the first column of the plate and we attended to successive three-fold dilutions with fresh RPMI containing 0.5% FBS. The last column (Column 12) of the plate contained drug-free medium. The cells were exposed to drugs for 30 min at 37 °C before being washed twice or thrice if the drugs were viscous or stained with RPMI (0.5% FBS). The cells were then put back in the incubator for 72 h. For each condition (type of molecule, concentration of molecule and cell line type), replicates (n = 4) were tested during the same experience. Each experience was repeated three times (N = 3), as previously described for a total period of four months.

The KBD and its three components as follows: (i) Nelumbo nucifera petals (NN), (ii) Piper nigrum fruits (BP), and (iii) the aerial part of Centella asiatica (CA) were provided by Chao Phya Abhaibhubejhr Hospital, Prachinburi Province, Thailand. The plants were identified by Benjawan Leenin, a chief of Traditional Knowledge Center, Chao Phya Abhaibhubejhr Hospital Foundation. The relative herbarium voucher specimens were deposited at the museum of Chao Phya Abhaibhubejhr Hospital with following voucher numbers: ABH15, ABH18, and ABH17, respectively. Acetylthiocholine iodide (ATCI), bovine serum albumin (BSA), beta amyloid 1-42 (Aβ_1–42), gallic acid, quercetin, β carotenoid, tacrine, trolox, N-acetyl cysteine (NAC), trypsine, fetal bovine serum (FBS), and Dulbecco's modified Eagle medium nutrient mixture F-12 (DMEM/F12) were purchased from Sigma-Aldrich (SM Chemical supplies Co., Ltd., Bangkok, Thailand), Merck (Merck, Bangkok, Thailand), Gibthai (GT Chemical supplies Co., Ltd., bangkok, Thailand), and Fluka (SM Chemical supplies Co., Ltd. ,Bangkok, Thailand).

To determine whether the cell death inducing component(s) were of protein nature, F. gastrosuis bacteria were pre-treated with pronase, trypsine or proteinase K (all at 1 mg/mL, 2 h, 37 °C) (Sigma-Aldrich) prior to incubation with MKN-7 and KYSE-450 cells. Bacteria were also pre-treated with paraformaldehyde (1%, 1 h, room temperature) to determine if cell death inducing agents were associated with the bacterial surface. Finally, F. gastrosuis bacteria and lysates were heat treated (100 °C, 10 min) to investigate their heat-lability. The percentages of viable, apoptotic and necrotic cells were flowcytometrically determined as described above. Negative as well as positive controls (i.e. untreated F. gastrosuis bacteria and lysates, respectively) were included. The viability of F. gastrosuis bacteria after protease, paraformaldehyde and heat treatment was verified by incubation on Columbia blood agar plates (37 °C, 48–72 h, anaerobic) after which bacterial growth was compared with that of untreated F. gastrosuis bacteria.

For RAW cell infection, 2 × 10⁴ cells per well were seeded in 12 wells plates (Corning) and grown for 4 days until ~70% confluence. The cydA reporter strain that also contained the integrated pMV-hsp60-mEos3.1 was grown to an OD600 of 0.8–1.5 and washed with DMEM + FBS (10%) prior to infection. Bacterial cells were subsequently added to a multiplicity of infection of 2. RAW cells were incubated at 33 °C After three hours, extracellular mycobacteria were removed by washing 2 times with PBS. Amikacin was added to a final concentration of 200 μg/mL for 3 hours to kill any remaining extracellular mycobacterial cells. Cell were then washed and BDQ was added at the indicated concentrations. At time points 24 hpi, 48 hpi, and 72 hpi RAW cells were harvested by addition of trypsine (Sigma). The trypsin was neutralized by addition of DMEM + FBS (10%) and cells were pelleted at 1400 rpm. Cells were subsequently fixated with 4% PFA for 30 minutes at room temperature, washed with PBS and analyzed by flow cytometry.

LC samples from patients or mice were removed, minced into small pieces, and digested in the digestion media containing 40 mg/dl collagenase (Sigma, USA) and 0.05% trypsine (Sigma-Aldrich) at 37 °C for 30 min. After the digestion, the cells that passed a 40 μm filter were pelleted and re-suspended in physiological solution, labeled with specific antibodies, and then used for flow cytometric analysis. Macrophages were analyzed by flow cytometry with specific fluorescence-conjugated antibodies (F4/80 and CD206--a M2 macrophage marker--, all from Becton-Dickinson Biosciences, San Jose, CA, USA). Data were collected on a FACSCalibur (Becton-Dickinson Biosciences) and analyzed using FlowJo software (Flowjo LLC, Ashland, OR, USA). The percentage expression of each marker on macrophages was determined by the percentage of positive events, as determined by the isotype-matched negative control.