Cell culture and stimulation
Human monocytic THP-1 cells (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific, MA, USA) and 1% penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA). Cells were incubated at 37°C in a mixture of 95% air and 5% CO2. To induce LPS tolerance, cells were stimulated with LPS (1st LPS, 200 ng/ml; Sigma-Aldrich, MO, USA) for 24 hours and then washed with phosphate buffered saline (PBS), followed by re-stimulation of LPS (2nd LPS, 200 ng/ml) for 2 hours. In the groups with treatment of MgSO4, MgSO4 (20 mM; Sigma-Aldrich, MO, USA) was added 24 hours before 1st LPS stimulation. The dose of MgSO4 was based on our previous study . THP-1 cells were randomly allocated into 6 groups (the PP, PL, LL, MPP, MPL and MLL group) illustrated in Figure 1. The experimental protocol of each group was illustrated in Figure 1. The PP and MPP group were control groups. LPS tolerance was induced in the LL and MLL group. The PL and MPL group received LPS stimulation without pre-exposure of LPS or development of tolerance.
The level of cell viability was measured using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, USA). In brief, cells were seeded in 96-well microplates, and incubated for 4 hours in a humidified atmosphere at 37 °C after MTT reagent was added to each well. Then, the absorbance was measured using a plate reader (Bio-Rad, Hercules, CA, USA) with a test wavelength at 570 nm and a reference wavelength at 630 nm. Cell viability was reported as the ratio of absorbance value in each group to that in the PP group.
Enzyme-linked immunosorbent assay (ELISA)
After treatment as previous description, the supernatant in each group was collected for assay of TNF-α. The concentration of TNF-α was quantified using the commercial ELISA kit of human TNF-α (R&D System, MN, USA) in accordance with the manufacturer’s protocol.
Assay of cell proliferation and morphology
To determine proliferation rate, THP-1 cells were seeded at a density of 104 cells/well in 96-well microplates. At 0, 24 or 48 hours (e.g., Day 0, Day 1 or Day 2) after end of 2nd LPS stimulation, viable cells in each group were determined using MTT assay after centrifugation. Then, ratio of proliferation was calculated as the ratio of absorbance value at each indicative time point relative to the absorbance value on Day 0. Finally, the growth curve in each group was plotted according to the average ratio of proliferation at each indicative time point. In addition, the ratio of proliferation in each group on Day 2 was compared relative to the PP group. To assay cell morphology, THP-1 cells were imaged using brightfield microscopy (Carl Zeiss, Inc., Oberkochen, Germany). For each group, we took at least 3 different fields under microscopy.
Measurement of cell adherence
Adherence of THP-1 cells is upregulated by LPS . To determine cell adherence, the adherent cells were collected by scratching, and stained using trypan blue. We used a hemocytometer to calculate the number of adherent cells. The percentage of adherence in each group was calculated relative to total seeded cells that represent 100%.
Transwell migration assay
A 24-well Transwell system (Corning®, NY, USA) with microporous polycarbonate membranes (10 µm thickness, 5 µm pores) was employed to measure cell migration capacity. In brief, THP-1 cells were seeded at a density of 106 cells/ml in upper inserts with fresh medium. FBS, as a chemoattractant, was added in lower inserts, instead of upper inserts. After migration at 37°C for 6 hours, non-migrated cells (on the upper side of membranes) were removed using cotton swabs, and migrated cells (on the bottom side of membranes) were stained with diamidino-2-phenylindole (DAPI, Pierce). The stained cells were observed under fluorescence microscopy. We took at least 3 different fields for each group, and calculated the number of migrated cells per field.
After treatment as previous description, the phagocytic activity of cells was measured using the Phagocytosis Assay Kit (Abcam, Cambridge, UK) according to the manufacture’s protocol. Briefly, zymosan was incubated with THP-1 cells at 370C, 5% CO2 for 3 hours. Then cells were harvested by centrifugation. After washing and suspension in Phagocytosis Assay Buffer, cells were analyzed by the plate reader at Ex/Em of 490/520 nm. The level of phagocytosis in each group was reported as the ratio to the PP group. Furthermore, another set of cells were seeded on the glass slides. After incubation with zymosan, cells were fixation and imaged under fluorescence microscopy. We took at least 3 different fields for each group.
Statistical analysis was performed using a commercial software package (SigmaStat for Windows; SPSS Science, Chicago, IL, USA). All data were presented as mean ± standard deviations, and analyzed by one-way analysis of variance in conjunction with Tukey’s post hoc test. A P-value < 0.05 was considered statistically significant.