Mar 27, 2025

Public workspacePreparation of Lipopolysaccharide for Intraperitoneal Injection

DOI
dx.doi.org/10.17504/protocols.io.14egn9y1ml5d/v1
  • Adrien Stanley1
  • 1Allen Institute for Neural Dynamics
  • Allen Institute for Neural Dynamics
Abbie Weaver
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DOI: dx.doi.org/10.17504/protocols.io.14egn9y1ml5d/v1
Protocol Citation: Adrien Stanley 2025. Preparation of Lipopolysaccharide for Intraperitoneal Injection. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn9y1ml5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 27, 2025
Last Modified: March 27, 2025
Protocol Integer ID: 119651
Keywords: Lipopolysaccharide, Intraperitoneal injections, LPS working solution
Abstract
This protocol describes the steps for preparing 20X stock and experimental solution of Lipopolysaccharide (LPS) intended for intraperitoneal injections in mice at a concentration of 0.25 mg/mL.

Guidelines
  • LPS can be given at dose 0.1 to 2.5 mg/kg in saline via intraperitoneal injection at a max volume of 0.5 mL, for a maximum of 1 day.
Materials

MaterialManufacturer/SupplierCatalog Number
5 mg LPS powderSigma-AldrichL3024-5MG
Pharmaceutical grade sterile saline (0.9%)Fisher ScientificNC9054335
20-200 µL PipetteRainin17014391
100-1000 µL PipetteRainin17014382
250 µL pipette tipsMettler-Toledo17014962
1000 µL pipette tipsMettler-Toledo17014966
1 mL syringeFisher Scientific309659
23-Gauge needle attachmentsBD BD305143
Low protein binding tubesFisher Scientific23-045-258
Caps for low protein binding tubesFisher Scientific22-038-265
Ethanol swabsUline13-680-63
ReagentLipopolysaccharides from Escherichia coli O111:B4Merck MilliporeSigma (Sigma-Aldrich)Catalog #L3024-5mg
Reagent0.9% SODIUM CHLOR. INJ.10MLThermo Fisher ScientificCatalog #NC9054335
ReagentPipet-Lite LTS Pipette L-200XLS RaininCatalog #17014391
ReagentLTS Pipette L-1000XLS Mettler ToledoCatalog #17014382
ReagentPipette Tips TR LTS 250µL S 960A/10Mettler ToledoCatalog #17014962
ReagentPipette Tips TR LTS 1000µL S 768A/8Mettler ToledoCatalog #17014966
Reagent1 ml tuberculin syringesBecton Dickinson (BD)Catalog #REF 309659
ReagentBD® Needle 3/4 in. single use, sterile, 23 GBecton Dickinson (BD)Catalog #305143
ReagentSimport™ Scientific Micrewtube™ Microcentrifuge Tubes with Low Adhesion SurfaceThermo Fisher ScientificCatalog #23-045-258
ReagentSimport™ Scientific Nonsterile Screw CapsThermo Fisher ScientificCatalog #22-038-265

Safety warnings
  • Proper PPE includes lab coat, eye protection, and disposable gloves. Report any accidental sticks to EHS.
  • When handling syringes with needles, always ensure needles are pointed away from you to avoid any accidental sticks.
  • Used needles and syringes should be disposed of in biohazard sharps container.
  • Lipopolysaccharide (LPS) is a potent immune stimulant and should be prepared in a fume hood and under sterile conditions with proper PPE.
Prepare 20X Stock Solution
Prepare 20X Stock Solution
1m
1m
Upon arrival of Amount5 mg Lipopolysaccharide (LPS) powder, in a fume hood dissolve in Amount1 mL of pharmaceutical grade sterile saline (0.9%) to make a Amount5 mg/mL stock solution.
Note
Note: If stock solution isn’t prepared upon arrival, store LPS powder at Temperature4 °C .


Attach 23-Gauge needle to 1 mL syringe.
Swab surface of sterile saline bottle septum with a 70% ethanol swab.
Withdraw Amount1 mL volume.

Eject saline in syringe into the bottle containing Amount5 mg LPS powder.

Mix the contents for ~Duration00:01:00 by inverting and shaking the bottle to ensure all powder on the bottle's interior surface is dissolved.

1m
Mix
Pipette Amount25 µL aliquots of stock solution in low protein binding tubes and store at Temperature-20 °C .
Note
Stock solutions can be stored at -20°C for approximately 2 years before expiring.


Remove label from the empty LPS bottle and discard bottle in biohazard waste.
Place label on clipboard that documents used chemicals in the lab.
Prepare 0.5 mg/mL LPS Working Solution for IP Injection
Prepare 0.5 mg/mL LPS Working Solution for IP Injection
1m
1m
At most Duration01:00:00 before the injection on the experimental day, dilute stock by 20X (to Amount0.25 mg/mL final concentration).

Rapidly thaw stock aliquot by holding fingers to the wall of the tube for about Duration00:01:00 .

1m
Add Amount475 µL of saline and mix by inverting the tube or by vortexing.

Pipetting
Mix
Leftover LPS should be discarded in a biohazard waste container.