TLR4; Lipopolysaccharides (LPS) primarily activate Toll-like receptor 4 (TLR4)/MD-2 complex, initiating innate immune responses [1][2]. Structural variations in core oligosaccharide regions (e.g., Escherichia coli R1-R4 vs. Salmonella) determine TLR4 binding affinity [2].

1. Please read references to determine experimental conditions, do not solely rely on the instructions shown beelow. It is recommended to determine the optimal experimental conditions by setting concentration and time gradients before formal experiments (the time and concentration of LPS treatment corresponding to the peaks of different inflammatory indicators vary).
2. To maintain the integrity of LPS, it is recommended to store LPS solutions in silanized containers. This is because LPS can adhere to plastic and certain types of glass, especially when the concentration is below 0.1 mg/mL. If the LPS concentration exceeds 1 mg/mL, this adsorption is relatively weak. It is recommended to prepare a stock solution with a concentration of ≥2 mg/mL, vortex thoroughly for more than 10 minutes, and use sonication if necessary to ensure full mixing and dissolution. If glass containers are used, ensure thorough mixing for at least 30 minutes before use to redissolve any LPS adsorbed on the tube wall.
3. LPS is a molecule that can form micelles of different sizes in solution and has an unfixed molecular weight. Stock solutions and working solutions can be directly prepared as mass concentrations (mg/mL, μg/mL, etc.).
4. A uniform turbid solution may be observed after LPS is dissolved in water or PBS. If filter sterilization is required, do not directly filter the stock solution. It is recommended to dilute it into a working solution first and then perform filter sterilization using a 0.22 μm filter membrane.

LPS is the main toxic component of Gram-negative bacteria, which can activate pathogen-associated molecular patterns (PAMPs) in the immune system and induce cells to secrete migrasomes. LPS can be recognized by TLR4 to activate the innate immune system, subsequently promoting the activation of NF-κB and the production of pro-inflammatory cytokines. It is commonly used in experiments involving the stimulation, activation, and differentiation of immune cells.
Different types of bacteria express LPS with different structures and bioactivities. LPS is generally divided into two configurations: rough (R) type and smooth (S) type. Among them, S-type LPS contains a typical three-part structure: O-antigen (a serotype-specific polysaccharide with repeating oligosaccharide units), core oligosaccharide (a C9-type non-repeating oligosaccharide), and Lipid A (the toxic component of LPS). R-type LPS does not contain O-antigen and is expressed as rough-type LPS. The lack of O-antigen can affect the process of LPS recognition by immune cells.
LPS expressed by the E. coli O55:B5 strain is a prototypical endotoxin often used as an endotoxin standard in LAL assays. Lipopolysaccharides from E. coli O55:B5 have high pyrogenicity and are commonly used for in vitro cell activation. Lipopolysaccharides from E. coli O55:B5 induce the secretion of pro-inflammatory cytokines in mouse macrophages.

1. Induction of Cellular Inflammatory Models
Pathogenic Mechanism
LPS binds to the TLR4-MD-2 complex on the cell surface, activating MyD88-dependent and MyD88-independent signaling pathways, promoting the nuclear translocation of transcription factors such as NF-κB, triggering the expression of inflammation-related genes, and leading to cellular inflammatory responses.

Specific Methods
Cell: Macrophages, tumor cells, glial cells and so on.
Administration: 0.1-10 μg/mL for 1-24 h

Note
(1) Before formal experiments, relevant references should be searched based on cell lines, LPS sources, etc., and concentration and time gradient screening should be performed to determine the optimal experimental protocol.
(2) LPS stimulation of cells does not necessarily cause cell death; therefore, it is not appropriate to determine the LPS modeling concentration and time solely by detecting cell viability. It is recommended to detect the expression and secretion of multiple inflammatory factors.
(3) During LPS stimulation of cells, cell morphological changes should be observed regularly. Excessively high concentrations may cause cytotoxicity, while excessively low concentrations may fail to effectively induce cellular damage.
(4) A certain concentration of DMSO can significantly inhibit LPS-induced inflammatory responses; it is recommended to dissolve LPS in PBS or ddH2O.
(5) In studies on the construction of in vitro inflammatory models, Lipopolysaccharides from E. coli O55:B5 and Lipopolysaccharides from E. coli O111:B4 are the most widely used LPS and are recommended!

Indicators of Successful Modeling
Inflammatory factors in supernatants or cells: Increased secretion and expression of IL-1β, IL-6, TNF-α, etc.
Increased NO release.
Inflammatory genes: Increased expression of iNOS, NF-κB, NLRP3, etc.
Lipopolysaccharides (LPS) primarily activate Toll-like receptor 4 (TLR4)/MD-2 complex, initiating innate immune responses [1][2]. Structural variations in core oligosaccharide regions (e.g., Escherichia coli R1-R4 vs. Salmonella) determine TLR4 binding affinity.

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Bacterial lipopolysaccharides (LPS) are unique and complex glycolipids that provide characteristic components of the outer membranes of Gram-negative bacteria. In LPS of the Enterobacteriaceae, the core oligosaccharide links a highly conserved lipid A to the antigenic O-polysaccharide. Structural diversity in the core oligosaccharide is limited by the constraints imposed by its essential role in outer membrane stability and provides a contrast to the hypervariable O-antigen. The genetics of core oligosaccharide biosynthesis in Salmonella and Escherichia coli K-12 have served as prototypes for studies on the LPS and lipo-oligosaccharides from a growing range of bacteria. However, despite the wealth of knowledge, there remains a number of unanswered questions, and direct experimental data are not yet available to define the precise mechanism of action of many gene products. Here we present a comparative analysis of the recently completed sequences of the major core oligosaccharide biosynthesis gene clusters from the five known core types in E. coli and the Ra core type of Salmonella enterica serovar Typhimurium and discuss advances in the understanding of the related biosynthetic pathways. Differences in these clusters reflect important structural variations in the outer core oligosaccharides and provide a basis for ascribing functions to the genes in these model clusters, whereas highly conserved regions within these clusters suggest a critical and unalterable function for the inner region of the core[2].

Please read references to determine experimental conditions, do not solely rely on the instructions shown beelow. It is recommended to determine the optimal experimental conditions (such as animal strains, age, dosage, frequency and duration, detection time and indicators, etc.) through preliminary experiments before formal experiments. Lipopolysaccharides (1.5 mg/kg; intraperitoneal injection; single administration) can induce nausea and hypothermia in mice, and trigger more severe and longer-lasting nausea responses in adult male mice.

1. Induction of Acute Lung Injury Model
Pathogenic Mechanism
LPS binds to cell surface receptors, activating inflammatory cells to release a large number of inflammatory mediators and triggering inflammatory responses. Excessive activation of inflammation leads to lung tissue damage, pulmonary edema, and pulmonary dysfunction, thereby establishing an acute lung injury model.
Specific Modeling Methods:
Mice: C57/BALB/c mice
Administration: 0.2-15 mg/kg via intratracheal administration

Note
(1) Before inducing animal models with LPS, relevant references should be searched based on experimental purposes, animal types, etc., and preliminary experiments should be conducted to determine the optimal experimental protocol.
(2) After LPS administration, the time points at which the content peaks of different inflammatory factors appear may be inconsistent. It is recommended to determine the experimental protocol based on references and select multiple time points for detection during preliminary experiments.
(3) LPS should be stored away from light and avoid repeated freeze-thaw cycles.

Indicators of Successful Modeling
Molecular changes: Increased secretion and expression of TNF-α, IL-6, IL-1β, etc., in BALF or lung tissue.
Histomorphological changes: After Hematoxylin-Eosin (HE) staining of lung tissue, intra-alveolar leukocyte infiltration, alveolar wall thickening, patchy hemorrhage, and interstitial edema are observed. The lung dry/wet weight ratio (D/W ratio) decreases.
Related product: Lipopolysaccharides, from E. coli O111:B4

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2. Induction of Cardiac Dysfunction/Myocarditis Model
Pathogenic Mechanism
Cardiac dysfunction (myocarditis) is a common complication of LPS-induced sepsis. LPS can activate inflammatory responses and oxidative stress, triggering myocardial cell damage, apoptosis, and cardiac fibrosis, thereby leading to cardiac dysfunction.
Specific Modeling Methods:
Mice: C57/ HsdWin:NMRI mice
Administration: 10 mg/kg via intraperitoneal injection (i.p.)
Indicators of Successful Modeling
Myocardial dysfunction: Significant reduction in left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF); significant increase in left ventricular end-systolic volume (LVESV) and left ventricular end-systolic diameter (LVESD).
Metabolic changes: Increased serum levels of CK-MB, LDH, AST, TNF-α, IL-6, IL-1β, etc.
Histomorphological changes: Disorderly arrangement of myocardial fibers, destruction of myocardial tissue with unclear contours, myocardial dissolution, interstitial edema, congestion, and inflammatory cell infiltration.

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Intraperitoneal LPS (1 mg/kg) in mice caused rapid neutrophil infiltration into kidneys, detected by Ly6G⁺ immunostaining (2.5-fold increase) within 6h. Urinary migrasome excretion peaked at 12h, correlating with podocyte injury markers (nephrin loss). Sex/age-dependent responses: Female and aged (18-month) mice showed 50% higher plasma IL-6 and TNF-α than males/young mice after LPS (0.5 mg/kg i.p.).

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Puberty is an important developmental event that is marked by the reorganizing and remodeling of the brain. Exposure to stress during this critical period of development can have enduring effects on both reproductive and non-reproductive behaviors. The purpose of this study was to investigate age and sex differences in immune response by examining sickness behavior, body temperature changes, and serum cytokine levels following an immune challenge. The effects of circulating gonadal hormones on age and sex differences in immune response were also examined. Results showed that male mice display more sickness behavior and greater fluctuations in body temperature following LPS treatment than female mice. Moreover, adult male mice display more sickness behavior and a greater drop in body temperature following LPS treatment compared to pubertal male mice. Following gonadectomy, pubertal and adult males displayed steeper and prolonged drops in body temperature compared to sham-operated counterparts. Gonadectomy did not eliminate sex differences in LPS-induced body temperature changes, suggesting that additional factors contribute to the observed differences. LPS treatment increased cytokine levels in all mice. However, the increase in pro-inflammatory cytokines was higher in adult compared to pubertal mice, while the increase in anti-inflammatory cytokines was greater in pubertal than in adult mice. Our findings contribute to a better understanding of age and sex differences in acute immune response following LPS treatment and possible mechanisms involved in the enduring alterations in behavior and brain function following pubertal exposure to LPS[2].

Human podocyte cell line (HPC) was used. HPCs were seeded onto culture plates and cultured in RPMI 1640 medium enriched with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin, and ITS. HPCs were cultured at 33°C and 5% CO2 for proliferation and were then shifted to 37°C and 5% CO2 for differentiation for 10–12 days. HPCs were incubated with or without different dose LPS, PAN, and HG for different time. HPCs were cultured with or without 50 μg/mL LPS, 75 μg/mL PAN, 60 mM HG, and different dose Rac-1 inhibitor (EHT 1864) for different time. In addition, HPCs were cultured with dynamin Inhibitor Dynasore or blebbistatin for 12 h[3].

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Podocyte injury model: Immortalized podocytes treated with LPS (0.1-10 μg/mL) for 24h. Migrasomes stained with anti-TSPAN4 antibody and quantified per cell using high-content imaging. Cytoskeletal changes assessed via phalloidin-F-actin staining [3]. Cytokine measurement: Peritoneal macrophages isolated from LPS-treated mice (0.5 mg/kg, 4h) and cultured for 24h. IL-6/TNF-α in supernatants measured by ELISA [4].

Animal/Disease Models:Female and male CD1 mice[2]
Doses: 1.5mg/kg
Route of Administration: Intraperitoneal injection, once
Experimental Results: Induced sickness behavior in all mice, but adult mice displayed more sickness than pubertal mice and adult males remained sick for a longer period of time than adult females. Caused a decrease in body temperature for all mice, but this decrease was greatest in adult males. Increased pro- and anti-inflammatory cytokines at various levels in pubertal and adult male and female mice, resulted in age and sex differences in cytokine concentrations following immune challenge. Only adult males and females treated with LPS displayed significantly more IL-6 than their saline controls, and pubertal males and females and adult females displayed significantly more IL-10 than their saline controls. All the mice displayed significantly more IL-12 and TNF-α than their saline controls.

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Podocyte injury model: C57BL/6 mice received intraperitoneal LPS (1 mg/kg in PBS). Urine collected at 0, 6, 12, 24h for migrasome isolation (ultracentrifugation). Kidneys harvested for histology [3]. Immune response study: Young (8-week) and aged (18-month) mice of both sexes injected with LPS (0.5 mg/kg i.p.). Blood collected at 1.5h for plasma cytokines; spleens weighed at 24h [4].

LPS (1 mg/kg i.p.) induced acute kidney injury in mice, evidenced by 2-fold increased urinary albumin/creatinine ratio at 24h [3]. Systemic toxicity included hypothermia (ΔT = -2.5°C), reduced locomotor activity, and 10% body weight loss at 24h post-injection (0.5 mg/kg) [4].

[1].Structural analysis of lipopolysaccharides from Gram-negative bacteria. Biochemistry (Mosc). 2010 Apr;75(4):383-404.

[2].Molecular basis for structural diversity in the core regions of the lipopolysaccharides of Escherichia coli and Salmonella enterica. Mol Microbiol. 1998 Oct;30(2):221-32.

[3].Podocyte-Released Migrasomes in Urine Serve as an Indicator for Early Podocyte Injury. Kidney Dis (Basel). 2020 Nov;6(6):422-433.

[4].Age and sex differences in immune response following LPS treatment in mice. Brain Behav Immun. 2016 Nov;58:327-337

LPS is an amphipathic glycolipid comprising lipid A (conserved), core oligosaccharide (variable), and O-antigen (strain-specific). It serves as a pathogen-associated molecular pattern (PAMP) triggering septic shock pathways [1][2]. Mechanism: Binds TLR4/MD-2 → MyD88-dependent NF-κB activation → proinflammatory cytokine storm [2][4]. Urinary migrasomes may serve as non-invasive biomarkers for early podocyte injury [3].

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This review covers data on composition and structure of lipid A, core, and O-polysaccharide of the known lipopolysaccharides from Gram-negative bacteria. The relationship between the structure and biological activity of lipid A is discussed. The data on roles of core and O-polysaccharide in biological activities of lipopolysaccharides are presented. The structural homology of some oligosaccharide sequences of lipopolysaccharides to gangliosides of human cell membranes is considered.[1]

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Background: Levels of urinary microvesicles, which are increased during various kidney injuries, have diagnostic potential for renal diseases. However, the significance of urinary microvesicles as a renal disease indicator is dampened by the difficulty to ascertain their cell source. Objectives: The aim of this study was to demonstrate that podocytes can release migrasomes, a unique class of microvesicle with size ranging between 400 and 2,000 nm, and the urine level of migrasomes may serve as novel non-invasive biomarker for early podocyte injury. Method: In this study, immunofluorescence labeling, electronic microscopy, nanosite, and sequential centrifugation were used to purify and analyze migrasomes. Results: Migrasomes released by podocytes differ from exosomes as they have different content and mechanism of release. Compared to podocytes, renal tubular cells secrete markedly less migrasomes. Moreover, secretion of migrasomes by human or murine podocytes was strongly augmented during podocyte injuries induced by LPS, puromycin amino nucleoside (PAN), or a high concentration of glucose (HG). LPS, PAN, or HG-induced podocyte migrasome release, however, was blocked by Rac-1 inhibitor. Strikingly, a higher level of podocyte migrasomes in urine was detected in mice with PAN-nephropathy than in control mice. In fact, increased urinary migrasome number was detected earlier than elevated proteinuria during PAN-nephropathy, suggesting that urinary migrasomes are a more sensitive podocyte injury indicator than proteinuria. Increased urinary migrasome number was also detected in diabetic nephropathy patients with proteinuria level <5.5 g/day. Conclusions: Our findings reveal that podocytes release the "injury-related" migrasomes during migration and provide urinary podocyte migrasome as a potential diagnostic marker for early podocyte injury.[3]

Typically exists as White to off-white solid at room temperature

LPS

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)