Hi fbs

Immortalized HAP 1 and MEF cell lines were used for this study.⁶ (link)^,8 (link) HAP 1 cells were grown Iscove’s Modified Dulbecco’s Medium (IMDM), supplemented with 10% Heat-Inactivated Fetal Bovine Serum (HI FBS; Life Technologies), 10 mL per L of Antibiotic Antimycotic Solution (Penicillin/Streptomycin/Amphoterichin B; Sigma Aldrich) and 2 mM L-Glutamine. MEF cells were grown in high-glucose Dulbecco’s Modified Eagle Medium (DMEM; Cytiva) supplemented with 10% HI FBS, 10 mL per L of Antibiotic Antimycotic Solution, and 1X Non-Essential Amino acids (NEAA; Lonza). HAP 1 Δ (c+δ) cell line that lacks c and δ-subunits of ATP synthase was used for the study of the role of ATP synthase in high-conductance PTP. MEF ANT Triple KO cell line lacking 3 ANT genes was used to study the role of ANT in high conductance PTP. MEF ANT Triple KO cells were grown in the same media as MEF WT cells with the addition of 1mM Sodium Pyruvate (Gibco) and 25mg/500mL Uridine (Sigma Aldrich). Cells were maintained in a humidified cell incubator, at 37°C under a 5% CO2 atmosphere.

Phagocytosis assays were performed as described previously [28 (link)]. Briefly, Human monocyte/macrophage cell line THP1 (ATCC, TIB-202) was maintained in RPMI-1640 medium (Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (HIFBS; GIBCO, Life Technologies, Grand Island, NY, USA), 1× glutaMAX (GIBCO, Life Technologies, Grand Island, NY, USA), and 1× antibiotic–antimycotic solution (GIBCO, Life Technologies, Grand Island, NY, USA) at 37 °C in a 5% CO₂ atmosphere.
(i) Fluorescence labeling of Klebsiella strains
Washed bacteria (ca. 10⁹ CFU) were suspended in 200 µL of PBS. After 2 h incubation at 37 °C, bacteria were harvested by centrifugation at 5000× g for 15 min at 4 °C and resuspended in PBS. For fluorescent labeling, bacteria were killed by UV for 1 h (Osram Germicidal Puritec HNS 30W G13, Saint Petersburg, Russia) and subsequently incubated with 1 mg/mL of fluorescein isothiocyanate (FITC, Thermo Scientific, Rockford, IL, USA) at 0.05 M carbonate/bicarbonate buffer (pH 9.5) at 37 °C for 30 min with gentle mixing in the dark. To remove the excess dye, FITC-conjugated bacteria were washed twice with ice-cold carbonate/bicarbonate buffer, resuspended in PBS, and stored at −80 °C until further use.
(ii) Uptake of FITC-labeled bacteria
First, fifty µL of UV-killed FITC-labeled bacteria in Hank’s Balanced Salt Solution Ca²⁺Mg²⁺ buffer (HBSS-Ca²⁺Mg²⁺; Lonza, Verviers, Belgium) supplemented with 1% HIFBS were added to 50 µL of non-adherent THP-1 cells (10⁷ cells/mL) in the same buffer to get an MOI of 100:1. After 2 h incubation at 37 °C in a humidified 5% CO₂ atmosphere, the cells were placed on ice to halt phagocytosis and then treated with 0.2 mg/mL of trypan blue solution to quench extracellular fluorescence. As a negative phagocytic control, each bacterial strain and phagocytic cell combination was also kept on ice to block the endocytic uptake of bacteria. For analysis by flow cytometry on FACS Calibur (Becton Dickinson, Franklin Lakes, NJ, USA), the samples were diluted 1:1 in ice-cold HBSS-Ca²⁺Mg²⁺ followed by being washed twice in ice-cold HBSS-Ca²⁺Mg²⁺. Each assay was performed at least three times in duplicate. The THP-1 effector cells were selected according to their forward and side scatter properties using THP-1 cells alone as a control. Data from 10,000 events (cells) per condition were collected and analyzed using Flowing Software (version 2.5.1).

Human hepatocyte (HepG2 cells, ATCC HB-8065) and cervical epithelial (HeLa, ATCC CCL-2) cell lines were grown in minimum essential medium (MEM) (+) Earle's salts and L-glutamine (Invitrogen), supplemented with 10% heat inactivated fetal bovine serum (HI-FBS; Lonza), 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). Mammalian cells were maintained at 37°C in 5% CO₂ atmosphere_. Cells were seeded in 24-well tissue culture plates and grown for 48 h (HepG2; 1×10⁵ cells/well) or 24 h (HeLa; 0.5×10⁵ cells/well) before infection.

Human fetal livers were obtained from elective abortions. Gestational age was determined by ultrasonic measurement of the diameter of the skull and ranged from 14 to 18 weeks. The use of this tissue was approved by the Medical Ethical Committee of the Academic Medical Center, Amsterdam, the Netherlands, subject to informed patient consent in compliance with the Helsinki Declaration. We isolated HFLCs on three independent occasions; in each case four fetal livers were pooled. Cells were isolated as described previously [14 (link)]. HFLCs were seeded in DMEM culture medium (Dulbecco's modified Eagle's medium, BioWhittaker) containing 10% heat-inactivated fetal bovine serum (HI-FBS, BioWhittaker), 2 mM L-glutamine (BioWhittaker), 1 μM dexamethason (Sigma), 10 μg/mL insulin, 5.5 μg/mL transferrin, 6.7 ng/mL selenium-X (ITS mix, Life Technology), 100 U/mL penicillin, 100 μg/mL streptomycin (penicillin/streptomycin mix, BioWhittaker) at a density of approximately 3*10⁵ cells/cm² in Primaria 6-well plates (BD Falcon). Clonal derivatives were obtained by limiting dilution. The selection procedure used and the functionality of the clonal derivatives are described elsewhere [14 (link)]. Near-confluent cultures were detached by 5 min incubation with 0.25% trypsin/0.03% EDTA (BioWhittaker) and split at 1:4 ratios. The number of population doublings (PD) was calculated as PD = log (N_f/N_i)/log 2, in which N_f is the final number of cells harvested and N_i is the number of cells initially seeded. No corrections were made for cells that did not re-attach after passaging, since their proportion was negligible. The period in which PD number progressed linear with culture time was used to calculate the PD time (T_PD).
Mature primary human hepatocytes were isolated from seven patients undergoing partial hepatectomy, because of metastatic carcinoma. The tumour free liver tissue used in each case for the hepatocyte isolation ranged between 2 to 10 grams. The procedure was approved by the Medical Ethical Committee of the Academic Medical Center subject to informed patient consent. The hepatocyte isolation method was adapted from the protocol described by Seglen [15 (link)] as previously described [14 (link)].
NKNT-3 cells were kindly donated by Prof. I. Fox, University of Nebraska, USA. The NKNT-3 cells were cultured on Primaria 6-well culture plates (BD Falcon) and in 75 cm2 culture flasks using CS-C complete serum free medium (Cell Systems Corporation) with 0.2 mg/ml hygromycin B (Invitrogen) and 1 U/ml penicillin/streptomycin (BioWhittaker). Cultures were passaged with a split ratio of 1:5 according to instructions for CS-C medium. Cre-mediated recombination to revert immortalization of NKNT-3 cells [4 (link)] was carried out by transduction of the adenoviral vector AxCANCre (Riken DNA Bank (Tsukuba Life Science Center, Japan) as described previously [5 (link)]. We analysed both reverted, hence transduced with AxCANCre and selected with G418, and unreverted i.e. untreated cells. HepG2 cells were obtained from ATCC (HB-8065) and cultured in Primaria tissue culture flasks in DMEM culture medium as described above for HFLCs.
All cultures were maintained at 37°C in a humidified atmosphere (95% air, 5% CO2) and the medium was changed every 2-3 days.