NO production was determined as reported by Yang et al. (2018) (link). RAW264.7 cells were solubilized in DMEM medium (5% FBS + 0.2% PS) at a concentration of 3 × 105 cells/ml, innoulcated in each 24-well multi-well plate and incubated in a 5% CO2 incubator at 37 °C for 24 h. The test sample was added to stimulate the cells (20 μg/ml; final conc.). The negative control was PBS, while the positive control was lipopolysaccharide (LPS) (10 g/ml) (Fujifilm Wako). Following activation, the medium was harvested, centrifuged at 12,000 rpm for 20 min and evaluated by Griess reaction, as reported by Baek et al. (2015) (link). A portion of each Griess reagent, medium supernatant sample, and 3.125–125 μg/ml sodium nitrite (NaNO3) standard solution was supplemented and incubated for 20 min. The absorbance at 550 nm was used as well as the nitrite concentration was quantify by standard curve.
Lipopolysaccharide
Manufactured by Fujifilm
Sourced in Japan
Lipopolysaccharide is a complex molecule found in the outer membrane of Gram-negative bacteria. It serves as a structural component and plays a role in the bacteria's interaction with their environment.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Gm csf, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Nacalai Tesque (2 mentions)
Interleukin 4 (il 4), by R&D Systems (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Nacalai Tesque (1 mentions)
Mem non essential amino acids solution, by Nacalai Tesque (1 mentions)
Hepes, by Nacalai Tesque (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Anti cd40 antibody, by BioLegend (1 mentions)
Rpmi 1640, by Fujifilm (1 mentions)
Dulbecco s phosphate buffered saline d pbs, by Fujifilm (1 mentions)
Celltracker orange, by Thermo Fisher Scientific (1 mentions)
Collagenase, by Fujifilm (1 mentions)
Sb431542, by Cayman Chemical (1 mentions)
Dulbecco modified eagle medium (dmem), by Fujifilm (1 mentions)
Fetal bovine serum (fbs), by Biosera (1 mentions)
C57bl 6 mice, by Japan SLC (1 mentions)
M1 macrophage generation medium dxf, by PromoCell (1 mentions)
Lipopolysaccharides (lps), by Fujifilm (1 mentions)
7 protocols using lipopolysaccharide
1
Nitric Oxide Production Assay in RAW264.7 Cells
2
Dendritic Cell Vaccination for Neoantigen-Specific CD8+ T Cells
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DCs were prepared as described previously [15 (link)]. Briefly, bone marrow cells from femurs and tibias were cultured in RPMI-1640 supplemented with 10% FBS, 10 mM HEPES (Nacalai Tesque), 1 mM sodium pyruvate (Nacalai Tesque), MEM Non-Essential Amino Acids Solution (Nacalai Tesque), 5 mM 2-mercaptoethanol (Sigma-Aldrich), 100 U/mL penicillin, 100 μg/mL streptomycin, and 20 ng/mL GM-CSF (PeproTech) for 8 days. DCs were stimulated with 1 μg/mL lipopolysaccharide (FUJIFILM Wako Pure Chemical Corporation) for 16 h and pulsed with peptides at 1 μg/mL for 2 h. For induction of neoantigen-specific CD8+ T cells, 1 × 106 neoepitope peptide-pulsed DCs were subcutaneously injected into the flank of mice twice biweekly. Two weeks after the second vaccination, the splenocytes were stimulated with the corresponding peptides and IFN-γ production was determined by intracellular cytokine staining. For evaluation of anti-tumor effects of neoepitope peptide-pulsed DC vaccines, mice were inoculated with 5 × 106 YTN16 cells on day 0 into the right flank. Neoepitope peptide-pulsed DCs were injected into the left flank on day 5. Tumor growth was monitored every 2 to 3 days.
3
Profiling Cytokine-Activated B Cells and Monocytes
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PBMCs from healthy donors or patients with chronic pulmonary GVHD reconstituted with 200 μL of RPMI 1640 (Nacalai Tesque, Kyoto, Japan) were aliquoted into 96-well plates (1 × 106 cells per well) and stimulated with IL-4 (200 IU/mL; R&D Systems) and anti-CD40 antibody (0.3 μg/mL; BioLegend, San Diego, CA) for 24 hours. The frequency of CD20+CD23+CD69+ cells, or the mean fluorescence intensity (MFI) of CD69+ in CD20+CD23+ B cells, was measured by using flow cytometry. Approval for analyzing patients’ peripheral blood samples was obtained from the Institutional Review Board of Kyoto University (G0697).
PBMCs reconstituted with 200 μL of RPMI 1640 were aliquoted into 96-well plates (1 × 106 cells per well) and stimulated with 1 μg/mL of lipopolysaccharide (Wako Pure Chemical Industries, Osaka, Japan) for 4 hours. Cells were fixed and permeabilized with FIX & PERM A/B buffers (Thermo Fisher Scientific). Intracellular TNF-α and surface staining were analyzed. Intracellular TNF-α was stained with allophycocyanin-conjugated mouse anti–TNF-α (MAb11, BioLegend MAb11), and the frequency of TNF-α–positive cells among CD68+ cells was determined according to flow cytometry.
PBMCs reconstituted with 200 μL of RPMI 1640 were aliquoted into 96-well plates (1 × 106 cells per well) and stimulated with 1 μg/mL of lipopolysaccharide (Wako Pure Chemical Industries, Osaka, Japan) for 4 hours. Cells were fixed and permeabilized with FIX & PERM A/B buffers (Thermo Fisher Scientific). Intracellular TNF-α and surface staining were analyzed. Intracellular TNF-α was stained with allophycocyanin-conjugated mouse anti–TNF-α (MAb11, BioLegend MAb11), and the frequency of TNF-α–positive cells among CD68+ cells was determined according to flow cytometry.
4
Preparation of Cell Culture Media
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Deionized water was generated
using a TW-300RU system (Nomura Micro Science, Kanagawa, Japan). Antibiotics
(penicillin–streptomycin, 100 folds, 168-23191) solution, trypsin
ethylenediaminetetraacetic acid (trypsin–EDTA, 10 folds, 208-17251)
solution without phenol red, nonessential amino acids (NEAAs, 100
folds, 139-15651), fluorescein (FL, 065-00252), lipopolysaccharide
(LPS, 125-05201), and Dulbecco’s phosphate-buffered saline
(D-PBS) (10 folds, 048-29805) were purchased from Fujifilm Wako Chemicals
(Osaka, Japan). Fetal bovine serum (FBS) (FB-1290/500) was purchased
from Biosera (Nuaille, France). The CellTracker Orange from Thermo
Fisher (Waltham, MA), Dulbecco’s modified Eagle’s medium
(DMEM) with phenol red (D6046), DMEM without phenol red (D4947), and
DMEM-high glucose (D6429) were purchased from Merck (Kenilworth, NJ).
using a TW-300RU system (Nomura Micro Science, Kanagawa, Japan). Antibiotics
(penicillin–streptomycin, 100 folds, 168-23191) solution, trypsin
ethylenediaminetetraacetic acid (trypsin–EDTA, 10 folds, 208-17251)
solution without phenol red, nonessential amino acids (NEAAs, 100
folds, 139-15651), fluorescein (FL, 065-00252), lipopolysaccharide
(LPS, 125-05201), and Dulbecco’s phosphate-buffered saline
(D-PBS) (10 folds, 048-29805) were purchased from Fujifilm Wako Chemicals
(Osaka, Japan). Fetal bovine serum (FBS) (FB-1290/500) was purchased
from Biosera (Nuaille, France). The CellTracker Orange from Thermo
Fisher (Waltham, MA), Dulbecco’s modified Eagle’s medium
(DMEM) with phenol red (D6046), DMEM without phenol red (D4947), and
DMEM-high glucose (D6429) were purchased from Merck (Kenilworth, NJ).
5
Lipopolysaccharide-Induced Vascular Smooth Muscle Cell Inflammation
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Primary mouse aortic VSMCs isolated from the aorta of wild‐type C57/BL6 mice (Japan SLC, Shizuoka, Japan) using collagenase (Wako Pure Chemical Industries) were incubated in DMEM (Wako Pure Chemical Industries) containing 10% fetal bovine serum (Biosera, Nuaillé, France) at 37°C and 5% CO2. After 12 hours of serum starvation, 1 ng/mL of lipopolysaccharide (Wako Pure Chemical Industries), with or without 100 ng/mL mouse recombinant osteoprotegerin, was added to the medium and incubated for 12 hours in the presence or absence of SB431542 (Cayman Chemicals, Ann Arbor, MI, USA)—a selective TGF‐β receptor I inhibitor that specifically blocks Smad signaling. The cells used for all experiments were subcultured for 3 to 6 passages.
6
Macrophage Phenotypic Polarization Protocol
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The murine macrophage-like cell line, J774A.1, was obtained from the Health Science Research Resources Bank (Tokyo, Japan) and cultured in Dulbecco's minimal essential medium (DMEM) with 10% fetal bovine serum (FCS). In addition, these cells were treated with 100 ng/mL of lipopolysaccharide (LPS; Wako, Tokyo, Japan) and 20 ng/mL of interferon (IFN) γ (Wako) for M1-like macrophages or 20 ng/mL of IL-4 (R&D Systems, Minneapolis, MN) for M2-like macrophages.
A human macrophage cell line was obtained from PromoCell GmbH (Heidelberg, Germany) and cultured in Monocyte Attachment Medium (PromoCell GmbH). In addition, for their differentiation into M1- and M2-like macrophages, cells were cultured in M1-Macrophage Generation Medium DXF (Promo-Cell GmbH) and M2-Macrophage Generation Medium DXF (PromoCell GmbH), respectively.
A human macrophage cell line was obtained from PromoCell GmbH (Heidelberg, Germany) and cultured in Monocyte Attachment Medium (PromoCell GmbH). In addition, for their differentiation into M1- and M2-like macrophages, cells were cultured in M1-Macrophage Generation Medium DXF (Promo-Cell GmbH) and M2-Macrophage Generation Medium DXF (PromoCell GmbH), respectively.
7
Dendritic Cell Preparation and Antigen Pulsing
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Bone marrow-derived dendritic cells (DCs) were prepared as described previously [15 (link)]. Briefly, bone marrow cells from femurs and tibias were cultured in RPMI-1640 (Nacalai Tesque) supplemented with 10% FBS, 12.5 mM HEPES, 5 × 10−5 M 2-mercaptoethanol, 1 × 10−5 M sodium pyruvate, 1% nonessential amino acids, 100 U/mL penicillin, 100 μg/mL streptomycin, and 20 ng/mL GM-CSF (PeproTech, Rocky Hill, NJ, USA) for 8 days. DCs were stimulated with 1 µg/mL lipopolysaccharide (FUJIFILM Wako, Osaka, Japan), 10 ng/mL GM-CSF, and 10 ng/mL interleukin-4 (PeproTech) overnight and pulsed with short peptide at 1 µg/mL for 2 h. DCs were pulsed with LPs (5 µg/mL) overnight and stimulated with lipopolysaccharide (1 µg/mL), GM-CSF (10 ng/mL) and interleukin-4 (10 ng/mL) for 4 h. To immunize mice, 1 × 106 DCs were subcutaneously injected into the flank.
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