Hek blue il 1β reporter cells

To quantify the IL-1β secretion, HEK-Blue IL-1β reporter cells (Invivogen) were used whereby binding of IL-1β to the surface receptor IL-1R1 results in the downstream activation of NF-kB and subsequent production of secreted embryonic alkaline phosphatase (SEAP) in a dose-dependent manner as previously described (Tsu et al., 2021a (link)). SEAP levels were detected using a colorimetric substrate assay, QUANTI-Blue (Invivogen), by measuring an increase in absorbance at OD655. Culture supernatant from treated or infected THP-1 cells was transferred to HEK-Blue IL-1β reporter cells plated in 96-well format in a total volume of 200 µL per well at 5×10⁵ cells/well. On the same plate, serial dilutions of recombinant human IL-1β (Peprotech) were added to generate a standard curve for each assay. After 24 hr, SEAP levels were assayed by adding 50 µL of the supernatant from HEK-Blue IL-1β reporter cells to 150 µL of QUANTI-Blue colorimetric substrate along with 0.25% Tween-20 to neutralize HIV virions in supernatant before readout. After incubation at 37°C for 15–30 min, absorbance at OD655 was measured on an Epoch Microplate Spectrophotometer (BioTek) and absolute levels of IL-1β were calculated relative to the standard curve.

All cell lines (HEK293T, HEK-Blue-IL-1β) are routinely tested for mycoplasma by PCR kit (ATCC, Manassas, VA) and kept at a low passage number to maintain less than 1 year since purchase, acquisition, or generation. HEK293T cells were obtained from ATCC (catalog # CRL-3216) and HEK-Blue-IL-1β cells were obtained from Invivogen (catalog # hkb-il1b) and all lines were verified by those sources and were grown in complete media containing DMEM (Gibco, Carlsbad, CA), 10% FBS, and appropriate antibiotics (Gibco, Carlsbad, CA). THP-1 cells were purchased from ATCC and grown in complete media containing RPMI (Gibco, Carlsbad, CA), 10% FBS, and 1% L-glutamine. For transient transfections, HEK293T cells were seeded the day prior to transfection in a 24-well plate (Genesee, El Cajon, CA) with 500 μl complete media. Cells were transiently transfected with 500 ng of total DNA and 1.5 μl of Transit X2 (Mirus Bio, Madison, WI) following the manufacturer’s protocol. HEK-Blue IL-1β reporter cells (Invivogen, San Diego, CA) were grown and assayed in 96-well plates (Genesee, El Cajon, CA).

All cell lines (HEK293T, HEK-Blue-IL-1β, and HaCaT) are routinely tested for mycoplasma by PCR kit (ATCC, Manassas, VA) and kept a low passage number to maintain less than one year since purchase, acquisition or generation. HEK293T cells were obtained from ATCC (catalog # CRL-3216), HEK-Blue-IL-1β cells were obtained from Invivogen (catalog # hkb-il1b) and HaCaT cells were obtained from the UC Berkeley Cell Culture Facility (https://bds.berkeley.edu/facilities/cell-culture) and all lines were verified by those sources. All cells were grown in complete media containing DMEM (Gibco, Carlsbad, CA), 10% FBS (Peak Serum, Wellington, CO), and appropriate antibiotics (Gibco, Carlsbad, CA). For transient transfections, HEK293T cells were seeded the day prior to transfection in a 24-well plate (Genesee, El Cajon, CA) with 500 µl complete media. Cells were transiently transfected with 500 ng of total DNA and 1.5 µl of Transit X2 (Mirus Bio, Madison, WI) following the manufacturer’s protocol. HEK-Blue IL-1β reporter cells (Invivogen, San Diego, CA) were grown and assayed in 96-well plates (Genesee, El Cajon, CA).