HEK-Blue hTLR4 Cell Culture and Stimulation Assay

HEK-Blue hTLR4 cells were cultured in complete growth medium: phenol red-free DMEM high glucose medium (Gibco) supplemented with 55-nM sodium pyruvate (Sigma-Aldrich), 10% heat-inactivated FBS (Gibco), 50 U/ml penicillin, 50 µg/ml streptomycin (Lonza, Walkersville, MD, USA), 2-mM l-glutamine (Gibco), 100 µg/ml Normocin (InvivoGen), and 1X HEK-Blue Selection (InvivoGen). All stimulation assays were performed on cells at passages between 3 and 15. To prepare cells for stimulation assays without detaching them from the plate, they were carefully rinsed once with ice-cold PBS to remove residual FBS. Then, 5 ml of pre-warmed PBS was added and dishes were incubated at 37°C for 2 minutes to promote cell detachment. Cells were fully detached from plates with gentle tapping, collected, and centrifuged at 52g for 5 minutes. Cell pellets were resuspended in complete growth medium without FBS at a final concentration of 1.4 × 10⁵ cells/ml and immediately used for stimulation assays.

To quantify the activation of TLR4 in HEK-Blue hTLR4 cells, experimental treatments with protein or peptide ligands were prepared in nuclease-free water (Ambion, Thermo Fisher Scientific), using protein LoBind tubes (Eppendorf, Hamburg, Germany) and low-binding tips (Sorenson BioScience, Inc., Murray, UT, USA). First, 20 µl of experimental treatments were added in duplicate or triplicate to a flat-bottom, 96-well plate (Falcon, Durham, NC, USA). Then, 180 µl of the cell suspension prepared at 1.4 × 10⁵ cells/ml was immediately added, and the plate was incubated for 24 hours at 37°C under 5% CO₂. Secreted alkaline phosphatase (SEAP) production was measured in each challenged well by transferring 20 µl of medium to a fresh 96-well plate containing 200 µl of pre-warmed Quanti-Blue detection medium (InvivoGen). The SEAP reporter gene is under the control of an IL-12 p40 minimal promoter fused to five nuclear factor kappa B (NF-κB) and activator protein 1 (AP-1) binding sites. Stimulation of these cells by TLR4 ligands activates NF-κB, which in turn stimulates SEAP production. The Quanti-Blue plate was developed at 37°C, 5% CO₂, for 3 hours followed by OD recordings at 630 nm using the Synergy2 microplate reader and Gen5 software. In order to normalize the results of each well to the number of cells, 20 µl of 10% Triton X-100 Surfact-Amps Detergent Solution (Thermo Fisher Scientific) was added to the wells of the challenged plate. The plate was placed on a shaker for 5 minutes to facilitate breakdown of cell membranes. Protein concentrations were measured using the Pierce BCA protein assay (Thermo Fisher Scientific) with standards prepared in DMEM following the manufacturer's microplate protocol. For plotting of hTLR4 activation, raw values of SEAP production were normalized to protein content obtained with the BCA assay. Mean OD values from untreated cells were subtracted as background from all other normalized values, with resulting values defined as specific activation. The percent specific activation was calculated relative to the maximum activation of TLR4 obtained with 50-nM S100A9.

In one set of experiments aimed to determine activation of TLR4, treatments were prepared with increasing concentrations of S100A9, CAP37, or CAP37-derived peptides. The final concentrations of S100A9 were 1, 5, 10, 50, 100, and 500 nM and 1 µM. The final concentrations of CAP37 were 100, 200, 300, 400, 500, 600, 700, 800, and 900 nM and 1 µM. All peptides were tested at final concentrations of 10 nM, 100 nM, 1 µM, 10 µM, and 100 µM.

In another set of experiments, we determined the effects of CAP37 and peptides on the activation of hTLR4 by S100A9. Experimental treatments were prepared by mixing an equal volume of S100A9 with CAP37 or its peptide. Mixtures were either added to plates immediately before adding cells or pre-incubated for 1 hour at room temperature prior to the addition of cells. In this set of experiments, S100A9 was used at a constant final concentration of 10 nM, which leads to 50% of the maximum TLR4 stimulation. This was to allow us to measure either an increase or a decrease in TLR4 activation in the presence of CAP37 and derived peptides. The final concentrations of CAP37 and peptides were 0.01, 0.1, 1.1, 11, 110, and 550 nM. A positive control of 10-nM S100A9 alone was included in all stimulation assays.