Raw 264.7 atcc tib 71

Dulbecco’s modified Eagle’s medium (DMEM), antibiotics (penicillin and streptomycin), and trypsin-ethylenediaminetetraacetic acid (EDTA) were purchased from Gibco BRL (Grand Island, NY, USA). Fetal bovine serum (FBS) was obtained from Biowest (Kansas City, MO, USA), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), LPS, 4-nitrophenyl n-acetyl-b-d-glucosaminide (p-NAG), and monoclonal anti-DNP-IgE were supplied by Sigma–Aldrich (St. Louis, MO, USA). DNP-BSA was procured from Invitrogen (Gaithersburg, MD, USA). Primary antibodies against p-p38, p38, p-JNK, JNK, p-ERK, ERK, p-Lyn, Lyn, p-Syk, Syk, p-PLCγ, PLCγ, and β-actin were obtained from Cell Signaling Technology (Danvers, MA, USA), and FcεRIγ, from LSBio (Seattle, WA, USA). SensiFAST SYBR No-ROX kit mix was purchased from Biolines (Seoul, Korea), and human filaggrin, AQP3, and HA ELISA kits were obtained from CUSABIO (Seoul, Korea). Rat basophilic leukemia (RBL-2H3, ATCC^® CRL-2256) and murine macrophage (RAW264.7, ATCC^® TIB-71) cells were procured from the American Type Culture Collection (ATCC), while human immortalized keratinocyte (HaCaT) cells were obtained from Prof. Lee of Chosun University in Korea.

Murine macrophages (RAW 264.7) (ATCC-TIB-71) were acquired from the American Type Culture Collection (ATCC) biobank. RAW 264.7 cells were grown in DMEM (Dulbecco’s Modified Eagle’s Medium) without phenol red, supplemented with 10% FBS, 1% antibiotic/antimycotic solution (Biowest, Riverside, MO, USA), and 1% sodium pyruvate (humidified atmosphere with 5% CO₂ at 37 °C). Cells were seeded in 96-well microplates (5 × 10⁴ cells/well) for cell viability and nitric oxide (NO) production assessment. For determination of interleukins’ levels, the cells were seeded in 12-well microplates (5 × 10⁵ cells/well).

Saponarin (purity > 98%) was obtained from Extrasynthese (Genay, France). Dulbecco’s modified Eagle’s medium (DMEM), antibiotics (penicillin and streptomycin), and trypsin-ethylenediaminetetraacetic acid (EDTA) were obtained from Gibco BRL (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Biowest (Kansas City, MO, USA), and lipopolysaccharide (LPS), monoclonal anti-DNP-IgE, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 4-nitrophenyl n-acetyl-b-d-glucosaminide (p-NAG) were obtained from Sigma-Aldrich (St. Louis, MO, USA). DNP-BSA was procured from Invitrogen (Carlsbad, CA, USA). Recombinant human TNF-α and IFN-γ were purchased from Peprotech (Rocky Hill, NJ, USA). Primary anti-bodies against ERK, JNK, p38, Lyn, Syk, PLCγ, STAT1, p-ERK, p-JNK, p-p38, p-STAT1, p-Lyn, p-Syk, p-PLCγ, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibody against TSLP was purchased from ABclonal (Woburn, MA, USA). Primary antibodies against FcεRIγ were purchased from LSBio (Seattle, WA, USA). The SensiFAST SYBR No-ROX kit mix was purchased from Biolines (Seoul, Korea). RAW264.7 (ATCC^® TIB-71) and RBL-2H3 (ATCC^® CRL-2256) cells were purchased from the American Type Culture Collection (ATCC). HaCaT cells were obtained from Byoung-Woo Lim of Konkuk University, Korea.

Vero E6 C1008 (ATCC CRL-1586) and RAW264.7 (ATCC TIB-71™) cells were purchased from American Type Culture Collection (Gaithersburg, MD, USA). THP1-Dual and A549-Dual cells were purchased from InvivoGen (San Diego, CA, USA). Viral stocks were produced by infecting Vero E6 cells with LCMV-Armstrong (ARM, strain 53b) or LCMV-WE (strain 54) at a low (0.01–0.001) MOI, resulting in stock titers of 1–5 × 10⁷ PFU/mL [59 (link)]. Infectious virus titration was performed in Vero E6 cells by standard plaque assay with 0.5% Avicel (FMC BioPolymer, Philadelphia, PA, USA) as previously described [60 (link)] with a limit of detection of approximately 80 PFU/mL. All work with infectious LCMV-WE was performed in BSL3 containment at either the NIH Regional Biosafety Laboratory run by the Center for Predictive Medicine on the University of Louisville campus or at the Galveston National Laboratory on the University of Texas Medical Branch campus according to protocols approved by their respective Institutional Biosafety Committees.

Standard lycorine (purity > 98%) was purchased from Chem Faces (Wuhan, China). Dimethyl sulfoxide (DMSO), phosphoric acid, triethylamine, and purified water were prepared by Milli Q® system from Millipore (Bedford, MA, USA). Acetonitrile HPLC grade was purchased from RCI Lab Scan (Bangkok, Thailand). Murine macrophage leukemia cell line (RAW 264.7: ATCC® TIB-71™) was purchased from American Type Culture Collection (ATCC®, VA, USA). Fetal bovine serum (FBS) was purchased from Gibco® (OK, USA). Dulbecco's Modified Eagle's Medium (DMEM), phosphate buffer saline (PBS), and penicillin-streptomycin (P/S) were purchased from Biochrom (MA, Germany). 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from Sigma (MO, USA).

The murine macrophage cell line RAW 264.7 (ATCC TIB-71) was obtained from the American Type Culture Collection (ATCC, Rockville, MD). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (FBS; Invitrogen) and a standard mixture of antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin, and 250 μg/ml amphotericin B) at 37 °C under an atmosphere of 5% CO₂. For cytotoxicity assays, RAW 264.7 cells were resuspended in DMEM supplemented with FBS (DMEM-10) and transferred into the wells of 24-well tissue culture plates. Wells were treated with 10 μg of M9 OMVs, 10 μg E. coli-derived OMVs, or PBS and incubated overnight. Treated RAW 264.7 cell supernatants were assayed for lactate dehydrogenase (LDH) release by using a CytoTox 96 nonradioactive cytotoxicity assay kit (Promega). Maximum release was achieved by lysis of monolayers with Triton X-100 at a final concentration of 1% (vol/vol). The LDH released by uninfected cells was designated the spontaneous release. Cytotoxicity was calculated as follows: percent cytotoxicity = (test LDH release − spontaneous release)/(maximal release − spontaneous release).