Protocols


Screen (pdf version)
ScreenTLR3 screen with IFN-gamma priming
Posted On03/17/2017 3:21 PM
AuthorLei Sun
Background

For more information about the TLR Signaling Screen, please click here.

Reagents and Solutions
Brewer’s thioglycolate medium, 4%
4% (w/v) Brewer’s thioglycolate medium powder (BBL Microbiology Systems, Cockeysville, MD) is added to distilled water pre-warmed to 37°C. Solution is autoclaved to sterilize and stored away from light.
 
PEC medium
Dulbecco’s modified eagle medium (Mediatech Inc., Herndon, VA)
10% (v/v) heat-inactivated fetal bovine serum
200 IU/mL penicillin
200 mg/mL streptomycin
 
MTT solution
2mg MTT (Sigma)/mL sterile PBS
 
IFNγ solution
Stock concentration: 100 μg/mL (R&D systems, 485-MI-100)
 
Poly(I:C) solution
Stock concentration: 2ug/ul (GE healthcare, 27-4732-01)
 
ELISA
TNFα ELISA kit (EBioscience)
Method
Peritoneal exudate cell (PEC) isolation
  • Three to four days prior to PEC isolation, 3 mL syringes filled with Brewer’s thioglycolate medium are used to inject mice intraperitoneally with 1.5mL through a 25-gauge needle.
  • Immediately prior to isolation, mice are anaesthetized under isofluorane vapour (2-5% v/v, 2% O2).
  • 5 mL syringes filled with sterile PBS are used to recover PECs by lavage through an 18-gauge needle. Once obtained, 1 ml exudate is added to 3 mL of PEC medium in a 96-deep well plate, and stored on ice.
  • Tubes containing exudate are centrifuged for 2 minutes at 1500 rpm in a tabletop centrifuge, and supernatant is replaced with 3 mL of PEC medium. Pelleted cells are resuspended by pipeting.
  • Plate 100 ul cells/ well (96 wells plate) for 3 days.
IFNγ priming
  • Prime PECs with 10ng/ml IFNγ for 4 hours
  • Wash twice with PBS
PEC activation with TLR ligands and TNF ELISA
  • Stimulate the PECs with 20 μg/ml poly(I:C) (for the TLR3 screen) for 4 hours.
  • Transfer the PEC supernatant to an ELISA plate and add MTT solution to the plate; incubate for 1 hour.
  • Add DMSO to the plate and perform an MTT assay. Read absorbance at 570 nm.