This product is derived from Escherichia coli 055:B5. Can be used to induce cells or induce disease in animal models such as inflammation and other related experiments. Lipopolysaccharides (LPS) is a characteristic component of the cell wall of Gram-negative bacteria, located in the outermost layer of the cell wall and exposed to the cell surface of non-capsular bacteria, which helps maintain the integrity of the outer cell membrane and protects the bacteria from damage by bile salts and lipid antibiotics. Structurally, lipopolysaccharides are composed of lipid A, core polysaccharide and O-polysaccharide side chains. Lipid A is the main component of bacterial endotoxin and determines its toxicity. The O-polysaccharide side chain is highly variable among different bacteria and determines the serotype of bacteria. LPS can induce a cascade of immune stimulation and toxic pathophysiological activities in the body.

Examples of using this product（for reference only）

In Vitro:

Cell（RAW264.7 cells；100 ng/mL LPS，24 h at 37 ℃）:

RAW264.7 cells were seeded in confocal dishes at a density of 1×10⁵ cells per dish.  After the culture of 24 h, cells were treated with 100 ng/mL LPS in different media, including a control medium, PACDFe extract, and PACDFe hydrogel extract supplemented with 1 μM LL-37 for another 24 h at 37 ℃.

Reference：

Hao Z, Liu G, Ren L, Liu J, Liu C, Yang T, Wu X, Zhang X, Yang L, Xia J, Li W. A Self-Healing Multifunctional Hydrogel System Accelerates Diabetic Wound Healing through Orchestrating Immunoinflammatory Microenvironment. ACS Appl Mater Interfaces. 2023 Apr 26;15(16):19847-19862. doi: 10.1021/acsami.2c23323. Epub 2023 Apr 12. PMID: 37042619.

Cell（RAW264.7 cells，1 μg/mL of LPS，48h）:

RAW264.7 cells were incubated in DMEM medium (containing 1 μg/mL of LPS) for 48 h to generate M1 macrophages, and were treated with 50 ng/mL of IL-4 for 24 h to obtain M2 macrophages. Then, the resulted macrophages were stained with CD86/PE (dilution 1:200) and CD206/FITC (dilution 1:100) double dye for 30 min. After being washed twice with PBS, cells were fixed with 4% paraformaldehyde for 10 min and then stained with 10 μg/mL DAPI for 10 min. The staining results were observed via confocal laser scanning microscope.

Reference：

Zhang X, Tang J, Li C, Lu Y, Cheng L, Liu J. A targeting black phosphorus nanoparticle based immune cells nano-regulator for photodynamic/photothermal and photo-immunotherapy. Bioact Mater. 2020 Sep 9;6(2):472-489. doi: 10.1016/j.bioactmat.2020.08.024. PMID: 32995674; PMCID: PMC7493086.

Cell（RAW264.7 cells，1 μg/mL of LPS，24h）:

For inflammatory experiments, the cells were seeded in a 12-well plate (4×10⁵ cells/mL for F4 subfraction and 2×10⁵ cells/mL for synthesized peptides, 1 mL/well) and allowed to adhere for 24 h. Afterward, the cells were pretreated for 1 h with the F4 subfraction dissolved in serum-free medium at 25?100 μg/mL. Thereafter, the cells were stimulated with 1 μg/mL LPS for 24 h. Finally, the media were collected, and nitric oxide (NO), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 levels were quantified.

Reference：

Hu S, Yuan J, Gao J, Wu Y, Meng X, Tong P, Chen H. Antioxidant and Anti-Inflammatory Potential of Peptides Derived from In Vitro

Gastrointestinal Digestion of Germinated and Heat-Treated Foxtail Millet (Setaria italica) Proteins. J Agric Food Chem. 2020 Sep 2;68(35):9415-9426. doi: 10.1021/acs.jafc.0c03732. Epub 2020 Aug 18. PMID: 32786864.

Cell（RAW264.7 cells，0.5 μg/mL of LPS，24h）:

An inflamma- tory model was established by lipopolysaccharide-stimulated mouse macrophage cell line RAW264.7, and the anti-inflammatory ability of ART was evaluated by ni- trite assay. All cells in this research were cultured in a DEME high sugar medium, in 5% CO2 incubator at 37℃. Mouse macrophages grown in log phase were seeded in a 96-well culture plate at a density of 1×106 cells/well in the DMEM medium. Fresh culture solu- tion containing only 0.5 μg/mL LPS (control group) or 0.5 μg/mL LPS + ART-12% PEG4000 (0.031 mg/mL, 0.063 mg/mL, 0.125 mg/mL, 0.248 mg/mL, 0.496 mg/mL, 0.982 mg/mL) was added to each well, and the culture was continued for 24 hours.

Reference：

Dai F, Bao Y, Li Z, Chen SH, Gao LZ, Liu ZL, Cheng L, Peng Y, Tong XL. Artemisinin is highly soluble in polyethylene Glycol 4000 and such solution has multiple biological effects. Acta Biochim Pol. 2020 May 18;67(2):203-211. doi: 10.18388/abp.2020_5190. PMID: 32421285.

Cell（RAW276.4；500 ng/mL Lipopolysaccharide/LPS；1 h）:

ROS and TNF-α were used as markers to characterize inflammatory response.   RAW276.4 macrophages were seeded on the tissue culture plates at a cell density of 100,000 cells/cm2 and cultured overnight, which were then stimulated with 500 ng mL? 1 of LPS for 1 h, followed by incubating with naked islets (50 IEQ) or 10% (v/v) of islets-laden microgels for 24 h. Intracellular ROS level was monitored by commfcially available assay kit (Beijing Solarbio Science & Technology Co., Ltd), and macrophages treated with 0.05 mg mL? 1 of Rosup were used as positive control.   As for TNF-α, stimulated macrophages were incubated with naked islets or islets-laden microgels for 12 h.

Reference：

Li H, Shang Y, Feng Q, Liu Y, Chen J, Dong H. A novel bioartificial pancreas fabricated via islets microencapsulation in anti-adhesive core-shell microgels and macroencapsulation in a hydrogel scaffold prevascularized In Vivo.Bioact Mater. 2023 Apr 20;27:362-376.   doi: 10.1016/j.bioactmat.2023.04.011.   PMID: 37180642;   PMCID: PMC10172916.

Cell（HepG2 cells，1 μg/mL LPS，12h）:

To monitor endogenous ClO?, HepG2 cells were treated with LPS(1 μg/mL) for 12 h and then coincubated with PMA (10 nmol/L) and the peptide@Ag/Au NCs solution at 37 ℃ for 1 h, followed by washing three times before imaging. In the control assay, HepG2 cells were treated with LPS (1 μg/mL) for 12 h and then incubated with PMA (10 nmol/L) for 1 h. The cells were subsequently cultured in medium containing uric acid (250 nmol/L) and DMSO (0.5%) for 15 min and then treated with the peptide@Ag/Au NCs solution at 37 ℃ for 1 h. The cells were washed three times with PBS to remove the unbound peptide@Ag/Au NCs and were then observed under a Nikon A1R MP multiphoton microscope with a 60× oil-immersion objective lens. The images of peptide@Ag/Au NCs were captured under excitation at409 nm.

Reference：

Jia M, Mi W, Guo S, Yang QZ, Jin Y, Shao N. Peptide-capped functionalized Ag/Au bimetal nanoclusters with enhanced red fluorescence for lysosome-targeted imaging of hypochlorite in living cells. Talanta. 2020 Aug 15;216:120926. doi: 10.1016/j.talanta.2020.120926. Epub 2020 Mar 14. PMID: 32456892.

T/B lymphocyte（Blymphocyte，1μg/ml LPS）

The proliferation of spleen T/B lymphocyte was also determined using an MTT assay as above. Briefly, 100 μl of cell suspension was seeded in a well of 96-well culture plates. After the addition of 50 μl Con A (16 μg/ml) or LPS (4 μg/ml) and 50μl COS-Se (80, 200, 400, 800 and 2000 μg/ml), the plates were incubated for 48 h at 37℃ in a 5% humidified CO2. The stimulation index (SI/%) was calculated using the following equation: SI (%) = Absorbance of COS-Se groups/Absorbance of Con A or LPS groups×100%.

Reference：

Jiang Z, Chi J, Li H, Wang Y, Liu W, Han B. Effect of chitosan oligosaccharide-conjugated selenium on improving immune function and blocking gastric cancer growth. Eur J Pharmacol. 2021 Jan 15;891:173673. doi: 10.1016/j.ejphar.2020.173673. Epub 2020 Oct 22. PMID: 33098836.

Lymphocyte（lymphocyte，15 μg/mL LPS）

Within 24 h of the last administration, the mice were sacrificed in a sterile environment. Spleen tissue was disrupted, and spleen cell suspensions were passed through sterile nylon mesh. Red blood cells were lysed by Erythrocyte Lysate (Solarbio, China). Cells were resuspended in RPMI 1640 medium (Solarbio, China) at the concentration of 5 x 106 cells/L. Blastogenic response of splenocytes to the mitogens, concanavalin A (ConA, Solarbio, China), and lipopolysaccharide (LPS, Solarbio, China), were assessed by CCK-8. Splenocytes suspension was incubated with ConA (10 μg/mL) or LPS (15 μg/mL) in 150μL RPMI 1640 medium containing 10% foetal bovine serum at 37 ℃ with 5% CO2. After incubation for 44 h, 10 lL CCK-8 was added to each well. After incubation for 2 h, the absorbance at 450 nm was measured by a microplate reader.

Reference：

Chen M, Fu Q, Song X, Muhammad A, Jia R, Zou Y, Yin L, Li L, He C, Ye G, Lv C, Liang X, Huang J, Cui M, Yin Z. Preparation of resveratrol dry suspension and its immunomodulatory and anti-inflammatory activity in mice. Pharm Biol. 2020 Dec;58(1):8-15. doi: 10.1080/13880209.2019.1699123. PMID: 31847682; PMCID: PMC6968662.

In Vivo:

Mice:

The LPS-induced acute lung inflammation modelMice were randomly divided into different groups: saline group, model group (LPS), DAS treatment groups and DAS prevention groups (delivered by either intratracheal instillation or intravenous injection, respectively). The LPS group was treated with 4 μg of LPS per mouse and the saline group was given the same volume of saline. The DAS prevention groups were administered with 25 mg/kg DAS at different time interval (e.g. 1 and 6 h) before LPS challenge. Mice were sacrificed at 6 h after LPS instillation and then the BALF were collected. The DAS treatment groups were administrated with LPS and the DAS (25 mg/kg) were delivered 1 h later. Mice were sacrificed at 6 h after LPS challenge and then the BALF were collected. The BALF was collected 3 times with 0.7 mL of saline each time, and the total volume of ~2 mL was recovered. The BALF samples were centrifuged (4 ℃, 1000 rpm, 15 min) and the supernatant was stored at ?20 ℃ for subsequent analysis.

Reference：

Wei-Ya C, Yuan-Song W, Chun-Yu L, Yu-Bin J, Fei-Fei Y, Yong-Hong L. Comparison of pulmonary availability and anti-inflammatory effect of dehydroandrographolide succinate via intratracheal and intravenous administration. Eur J Pharm Sci. 2020 Apr 30;147:105290. doi: 10.1016/j.ejps.2020.105290. Epub 2020 Mar 2. PMID: 32135270.

Mice（Female wild-type C57BL/6 mice， 2 mg/kg/every 3 days LPS, i.p）:

In other experiments, mice were serially treated with recombinant CCL2 (rCCL2) (10 μg/kg/every 3 days, i.p.), Toll-like receptor 4 (TLR4) specific ligand (lipopolysaccharide (LPS) -EB Ultrapure, 2 mg/kg/every 3 days, i.p.), LPS (2 mg/kg/every 3 days, i.p.; Solarbio), TLR4 inhibitor (TAK-242, 10 mg/kg/every 3 days, i.p.) or cyclooxygenase-2 (COX-2) inhibitor (Celecoxib, 10 mg/kg/every 3 days, intragastric administration). Polyp or tumour load was determined as the sum of the size of all colon polyps/tumours presented per mouse.

Reference：

Yang Y, Li L, Xu C, Wang Y, Wang Z, Chen M, Jiang Z, Pan J, Yang C, Li X, Song K, Yan J, Xie W, Wu X, Chen Z, Yuan Y, Zheng S, Yan J, Huang J, Qiu F. Cross-talk between the gut microbiota and monocyte-like macrophages mediates an inflammatory response to promote colitis-associated tumourigenesis. Gut. 2020 Oct 29;70(8):1495–506. doi: 10.1136/gutjnl-2020-320777. Epub ahead of print. PMID: 33122176; PMCID: PMC8292576.

Zebrafish larvae（Tg (Lyz:DsRed) transgenic line斑马鱼幼虫）:

After exposure to 72 hpf, the Tg (Lyz:DsRed) transgenic line was treated with LPS and cyhalofop-butyl together up to 96 hpf, and then the number of macrophages was observed by neutral red staining to detect the inflammatory response. The tail fins of larvae were cut under a stereomicroscope (Leica S6, Germany) after 96 h of exposure and anesthetized with 0.16% Tricaine.

Reference：

Cheng B, Zou L, Zhang H, Cao Z, Liao X, Shen T, Xiong G, Xiao J, Liu H, Lu H. Effects of cyhalofop-butyl on the developmental toxicity and immunotoxicity in zebrafish (Danio rerio). Chemosphere. 2021 Jan;263:127849. doi: 10.1016/j.chemosphere.2020.127849. Epub 2020 Aug 22. PMID: 33297003.