Human tlr1 9 agonist kit

To analyze the transcriptional response of JFB organoids to stimulation with pathogen-associated molecular patterns, organoids were trypsinized and then re-embedded into Matrigel in the presence of the following TLR agonists (Human TLR1-9 agonist kit, InvivoGen): TLR1, Pam3CSK4, 1 µg/mL; TLR2, heat-killed Listeria monocytogenes (10⁸/mL); TLR3, low molecular weight poly I:C, 10 µg/mL; TLR7, imiquimod, 1 µg/mL; TLR9, ODN2006, 5 µM. Alternatively, organoids were treated with UV-inactivated SARS-CoV-2⁹⁰ (10 µg/mL). After 48 h, organoids were lysed in TRI Reagent (Sigma) and processed for RNA isolation and RT-PCR.

To analyze transcriptional response of JFB organoids to stimulation with pathogen-associated molecular patterns, organoids were trypsinized and then re-embedded into Matrigel in the presence of the following TLR agonists (Human TLR1-9 agonist kit, InvivoGen): TLR1, Pam3CSK4, 1 μg/mL; TLR2, heat-killed Listeria monocytogenes (10⁸/mL); TLR3, low molecular weight poly I:C, 10 μg/mL; TLR7, imiquimod, 1 μg/mL; TLR9, ODN2006, 5 μM. Alternatively, organoids were treated with UV-inactivated SARS-CoV-2 ⁷⁶ (10 μg/mL). After 48 h, Organoids were lysed in TRI Reagent (Sigma) and processed for RNA isolation and RT-PCR.

TLR ligands (Human TLR1–9 Agonist kit, Invivogen, Cat. # tlrl-kithw) were prepared as recommended by the manufacturer prior to addition to cell cultures. Safe, non-toxic doses of these TLR agonists were chosen by the propidium iodide (PI) exclusion method [described elsewhere, counting stained (dead) cells with a Cellometer^® Vision automatic cell counter (Nexcelom Bioscience, MA)], after experimentation with THP-1/HIV (HA3) cells for further treatment with the rest of the cell lines tested.
For testing NF-κB dependence of TLR ligands-mediated HIV reactivation, cells were pre-treated for 2 h with either 100 µM of IKKγ NEMO binding domain inhibitory peptide, or equivalent amount of the control peptide (Imgenex), prior to incubation with indicated doses of TLR ligands for 16 h. Quantification of GFP⁺ cells by flow cytometry followed.
In general, assays on suspension cells (THP-1, U937, SC, and Jurkat) were carried out at a density of 1 × 10⁶ cells per mL, in 96-well plates in a volume of 100 µL. Assays on microglial cells (attached) were carried out in 24-well plates containing 1 × 10⁵ cells per well plated at least 8 h prior to treatments. Cell culture maintenance was carried out at 37 °C in 5% CO₂, and treatments were performed under the same conditions for 16 h prior to evaluation of viral reactivation by flow cytometry and/or fluorescence microscopy.

SZ95 cells are an immortalized human sebocyte cell line generated from the face of an 87-year-old female and transformed with Simian virus 40 (RRID:CVCL_9803). These cells were previously obtained from Cristos Zouboulis (Harris et al., 2019 (link)). SZ95 sebocytes were maintained in Sebomed Basal Medium (Fisher Scientific NC9711618) supplemented with 10% fetal bovine serum (GeminiBio 100-106), 5 ng/ml human epidermal growth factor (Thermo Fisher PHG0313), and 1% antibiotic–antimycotic (Gibco 15240062). Cells were cultured in 5% CO₂ incubator at 37°C. Cells were stimulated with 1 μg/ml LPS (Sigma L4524). Sixteen hours poststimulation cells were harvested and analyzed as described below. For TLR agonists treatment, reagents in human TLR1-9 Agonist kit (InvivoGen, tlrl-kit1hw) were diluted to working concentration in PBS. 100 ng/ml Pam3CSK4, 1 × 10⁸ cells/ml HKLM, 500 ng/ml Poly (I:C), 500 ng/ml Flagellin, 100 ng/ml FSL-1, and 1 μg/ml Imiquimod were used for stimulation of SZ95 cells. For heat-inactivated bacteria treatment, bacteria were grown to mid-logarithmic phase, spun down, washed, and resuspended in PBS. Bacteria were heat-inactivated by incubation at 95^oC for 20 min. Then 1 × 10⁸ cells/ml were used for each treatment.

Following differentiation, the multinucleated myotubes were exposed to 50 and 500 ng/ml Pam3CSK4 (TLR1/2L), 10⁶, 10⁷ and 10⁸ cells/ml heat-killed Listeria monocytogenes (HKLM) (TLR2L), 10 ng/ml, 1 and 50 µg/ml Poly (I:C) (TLR3L), 50 pg/ml, 10, 50, 100 and 500 ng/ml, 1, 2 and 10 µg/ml LPS (TLR4L), 50 pg/ml, 10 and 100 ng/ml flagellin (TLR5L) and 1 ng/ml and 1 µg/ml FSL-1 (TLR6L) (from the Human TLR 1–9 agonist kit from InvivoGen) in the differentiation media as described above prior to the substrate uptake and oxidation assays. The ligand concentrations were based on either the manufacturer’s suggested range or as previously described in published studies^{29 (link)}.

In order to analyze the effect of different Toll-like receptor agonists on the secretion of IL-6 and sIL-6R into the supernatant, 5 × 10⁵ PBMCs in 1 ml serum-free RPMI (Sigma-Aldrich, St. Louis, MO, USA) were incubated with a panel of Toll-like receptor agonists (Pam3CSK4 (1 µg/ml), Heat Killed Listeria monocytogenes (HKLM, 10⁸ cells/ml), Poly(I:C) (25 µg/ml), Poly(I:C) LMW (25 µg/ml), LPS K12 (5 µg/ml), Flagellin (1 µg/ml), FSL-1 (1 µg/ml), Imiquimod (5 µg/ml), ssRNA40 (5 µg/ml) (Human TLR1-9 Agonist kit, Invivogen, San Diego, CA, USA) for 24 h at 37 °C and 5% CO₂. Afterwards, the supernatant was collected and further analyzed.