RAW 264.7 macrophage-like cells were purchased from the American Type Culture Collection (No TIB-71; American Type Culture Collection [ATCC], Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle medium-high glucose media (DMEM-HG) (HyClone Laboratories Inc, Logan, UT, USA), 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin, then incubated in 5% CO2 at 37°C and 100% humidity until ~80% confluent. Cells were detached from the culture plates using a cell scraper, then centrifuged at 1500 rpm for 5 minutes and reseeded in four separate culture plates at a density of 5.0 × 105
cells/mL, in FBS-starved media (DMEM-HG with 1% penicillin-streptomycin only). After a 16-hour starvation period, the cells were either induced with LPS or left as controls. The cultured RAW cells were challenged for 3 hours (acute-phase inflammatory response was optimized at 3 hours [data not shown]) with LPS to initiate an inflammatory response. After the 3-hour LPS induction period, separate cell plates were exposed to PEMF at different frequencies for 1 hour.
In order to obtain the optimal dosage concentration for the inflammatory response, a bacterial LPS pellet from Escherichia coli 055:B5 (Sigma-Aldrich, St Louis, MO, USA) was resuspended in Dulbecco’s sterile phosphate-buffered saline at a concentration of 1 mg/mL and applied to the cell solution at serial concentrations of 1.5 μg/mL, 3 μg/mL, 5 μg/mL, 8 μg/mL, and 10 μg/mL, and then incubated for 3 hours in 5% CO2 at 37°C and 100% humidity. NFkB assay (NFkB (p65) Transcription Factor Assay Kit, catalog #89859; Thermo Fisher Scientific, Waltham, MA, USA) was used to test NFkB activation in the inflammatory response. No significant cell death was seen after testing the LPS-induced cell population using the Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay (MTS) (catalog #G3580; Promega Corp, Fitchburg, WI, USA) (data not shown).
A PEMF device (), consisting of a pair of 14 cm diameter coils placed 7 cm apart (), was constructed with 2400 turns of copper wire, in the form of a Helmholtz coil. The coils were driven by an alternating current power supply with adjustable frequency and amplitude. From the coil center inside the 4 cm coil region, the uniform field strength was measured to be approximately 40 Gauss (). By increasing or decreasing the voltage, the coils were able to emit various frequencies in the sub-ELF-EMF range of 5 Hz to 30 Hz. Each coil carried a 50% duty cycle square wave in the same direction (). The PEMF was characterized using a Gauss meter (Sypris Model 5180; Pacific Scientific-OECO, Milwaukie, OR, USA) to determine the average surface field density of ~40 Gauss. The frequency was determined using an oscilloscope (Model TDS 20248; Tektronix Inc, Beaverton, OR, USA). Experiments were performed under ambient conditions. After exposure to PEMF, a live/dead cell assay was performed using calcein acetomethoxy (catalog # C697959; Life Technologies, Carlsbad, CA, USA) showing no significant change in cell viability (data not shown). Measurements were taken using a Spectramax M5 plate reader (Molecular Devices LLC, Sunnyvale, CA, USA), at 428 nm excitation level and 520 nm emission level. Control samples were kept in the same conditions without exposure to PEMF. A custom built thermocouple was used to ensure a consistent 37°C temperature during this process. Background EMF was measured to average the same as Earth’s ~0.05 Gauss.
(A) The PEMF device was designed as a Helmholtz coil to hold a 6-well cell plate. (B) Two coils spaced half the distance of the diameter of the coils emitted a (C) uniform magnetic field. (D) The 5 Hz pulsed waveform.
Four experimental groups were set up as follows: (1) RAW macrophages were used as the negative control; (2) RAW macrophages induced with LPS were used as the positive control; (3) RAW macrophages exposed to a magnetic field were one treatment group; and (4) RAW macrophages induced with LPS and exposed to a magnetic field were the other treatment group. For all experiments, four polystyrene tissue-cultured treated 6-well plates were placed directly in the center of the magnetic field with cells only in the four outer wells (). The two center wells were empty to assure the uniform cell exposure to PEMF of the four outer wells. The control groups were placed in identical but separate conditions away from PEMF exposure. Three independent trials were run at each of the various frequencies (5.1 Hz, 7.8 Hz, 10.8 Hz, 15.6 Hz, 20.8 Hz, 23.4 Hz, and 30 Hz). After 1 hour of continuous PEMF exposure at each of these frequencies, the supernatant was extracted from the cell plates, and a multianalyte enzyme-linked immunosorbent assay (ELISA, catalog # SEM-03113A, SA Biosciences, Valencia, CA, USA) was used to test for TNF-α production. Cell lysate from the same trials was used for detection of NFkB. Separate trials were run to test for expression of A20, after discovering the significance of the 5.1 Hz frequency.
Cells were plated in 6-well plates in the four outer wells only, to assure uniform exposure to the magnetic field.
Detection of cell signaling
NFkB measurements were obtained using a NFkB assay. The NFkB transcription factor assay used detects active NFkB in both the primary antibody p65 and a secondary horseradish peroxidase (HRP) conjugated antibody. The measurement of TNF-α production was obtained using a Mouse Inflammatory ELISA (catalog #SEM-03113 A; Qiagen, Venlo, The Netherlands), which measures an individual protein analyte using a standard sandwich ELISA technique. The ELISA analyzed TNF-α using a conventional protocol, where the capture antibodies bind to specific proteins. The color development was read at an absorbance level of 450 nm. Since possible turbidity (which goes uncorrected in an 450 nm reading ELISA) may yield an artificially high signal, we subtracted the 450 nm reading from the 570 nm reading, to correct for any minor optical imperfections in the ELISA plate.
To extract and purify ribonucleic acid (RNA), 350 μL of the cell lysate samples were processed with the RNeasy Plus Mini Kit (catalog #74134; Qiagen) according to the manufacturer’s instructions, with slight modification for elution volume. RNA quality and concentration was measured using a Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific). Purified RNA was eluted in 50 μL elution buffer and stored at −80°C.
Complementary deoxyribonucleic acid (cDNA) synthesis
cDNA was generated from RNA extraction, using a master mix of 2 μL 10X random primers, 2 μL 10X reverse transcriptase (RT) buffer, 0.8 μL 10 mM deoxyribonucleotide triphosphate (dNTP), 10 μL of RNA and distilled water, and then incubated in a Peltier Thermal Cycler (PTC-200 DNA Engine®, Bio-Rad Laboratories Inc, Hercules CA, USA). The solution was then added to 1 μL Superscript® II (catalog #18064-014; Life Technologies) and incubated at 25°C for 10 minutes, then at 42°C for 50 minutes, then at 70°C for 15 minutes, and then stored at −20°C until qRT-PCR was conducted. The primers for the measurement of the steady state levels of mRNA of the A20/TNFAIP3 gene product were forward: 5′-GCA AGG CTG GGA CCA CG - 3′ and reverse: 5′ TTG GGT AAG TTA GCT TCA TCC AATT -3′ (database accession number 7427750; Eurofins MWG Operon, Huntsville, AL, USA).
A20 gene expression was quantified using cDNA from cell samples by qRT-PCR. A Sequence Detection System 7300 (Life Technologies) was used for amplification and specific sequence detection. Forward and reverse PCR primers were used in a master mix of 8 μL of distilled water, 10 μL SYBR green (Life Technologies), 0.5 μL each of forward primer (GCA AGG CTG GGA CCA CG) and reverse primer (TTG GGT AAG TTA GCT TCA TCC AATT), and 1 μL cDNA for a total volume of 20 μL. This master mix was used with both glyceraldehyde phosphate dehydrogenase (GAP DH), which was used as the endogenous gene; and A20, the target gene. Cycling parameters were: 2 minutes, 94°C; 30 sec, 94°C; 30 sec, 55°C and 30 sec, 72°C, for 34 times; and finally, 10 minutes, 72°C. GAP DH primers were purchased from Applied Biosystems/Life Technologies.