shRNA

Transducing Bone Marrow Derived Macrophages Protocol

Product No. SHM01

Protocol courtesy of Sigma® MISSION® RNAi Team, Pat Blanner, Chris Bauer, Jason Books, Alex Hromockyj; Inflammation Pathways Group within Pfizer Global Research and Development - St. Louis.

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Materials

  • Male DBA mice
  • 10 cc syringe
  • 30 gauge needle
  • Swinging Bucket Centrifuge
  • T-75 culture flasks (C7231)
  • T-150 culture flasks (CLS430825)
  • 60 mm Petri dishes (CLS430166)
  • 96-well plates (CLS3595)
  • ExpressMag Super Magnetic Kit (SHM01)
  • MEM alpha media without phenol red supplemented with L-glutamie
  • Growth Medium - MEM alpha media without phenol red supplemented with: L-glutamine, 10% FBS, antimycotics, antibiotics, 20 ng/ml M-CSF (M9170)
  • Selection Medium – Growth Medium supplemented with 3 µg/ml puromycin (P9620)
  • DPBS DI - Dulbecco’s Phosphate Buffered Saline (D8662) supplemented with 1.5 mg/ml Dispase I (D4818) prepared fresh each usage
  • LPS (L4641)
  • Tissue culture incubator—37 °C, 5% CO2, 100% relative humidity
  • Tissue culture laminar flow hood—only open optimization plate in tissue culture hood
  • Centrifuge with swinging bucket rotor
  • MISSION lentivirus (SHC002V in example)
  • Sterile cell scrapers

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Protocol

Day 0
  0.1   Determine the number of femurs needed we estimate approximately 1.5 million BMDM per femur by the end of the protocol.
  0.2   Make an ice-cold solution 20 ml of MEM alpha and maintain on ice.
  0.3   Pre-warm growth media to 37 °C.
  0.4   Euthanize mice as per protocols approved at your institution.
  0.5   Remove femurs from the mice and immediately place in ice cold MEM alpha.
  0.6   Using a 10 cc needle and 30 ml syringe flush bone marrow from femurs into 60 mm Petri dish.
  0.7   Gently pipette the bone marrow to disrupt.
  0.8   Centrifuge at room temperature for 5 minutes at 1,000 RPM.
  0.9   Remove supernatant without disrupting cell pellet.
  0.10   Immediately add 20 ml of pre-warmed growth media and gently resuspend by pipetting.
  0.11   Transfer to T-75 and incubate overnight in tissue culture incubator.
 
Day 1
  1.1   Pre-warm growth media.
  1.2   Transfer to sterile 50 ml conical.
  1.3   Centrifuge at room temperature for 5 minutes at 1,000 RPM.
  1.4   Resuspend pellet in 50 ml of growth media.
  1.5   Transfer to T-150
  1.6   Return to tissue culture incubator.
 
 
Day 3
  3.1   Pre-warm growth media.
  3.2   Replace media on cells
  3.3   Return flask to tissue culture incubator.
 
Day 5
  5.1   Pre-warm growth media.
  5.2   Remove media from flask.
  5.3   Add 10 ml of DPBS DI per flask and remove cells with gentle scraping.
  5.4   Cells were counted.
  5.5   Centrifuge at room temperature for 5 minutes at 1,000 RPM.
  5.6   Resuspend cells in growth media at a concentration of 1x106 cells/ml
  5.7   Add 100 μl of cells in media to each well required in a 96-well plate
 
Day 6
  6.1   Thaw MISSION lentivirus
  6.2   Calculate volume of virus (Vv) in μl per well for an MOI of 8.
  6.3   Utilizing the following formula calculate the volume of DMEM (Dv) needed per well to bind virus to beads then add DMEM to empty tube. Dv=50-Vv-1.5.
  6.4   Add 1.5 μl of ExpressMag beads (SHM03) per well to the DMEM.
  6.5   Gently mix by pipetting
  6.6   Add 50 μl of the mixture per well.
  6.7   Place 96-well plate on supermagnetic plate for 15 minutes at room temperature.
  6.8   Return cells to tissue culture incubator over night.
 
Day 7
  7.1   Pre-warm selection medium.
  7.2   Remove media from wells in 96-well plate.
  7.3   Replace with 100 µl per well of selection medium.
  7.4   Return to tissue culture incubator.
 
Day 9
  9.1   Repeat 7.1 - 7.4.
 
Day 11
  11.1   Perform desired assay.

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