Mouse intestinal epithelial cell strip

 Mouse intestinal epithelial cell strip

General notes

• It is recommended to wear a lab coat throughout the procedure
• Ensure you have strip buffer before beginning – prepare using sterile reagents and technique, once made can be used at the bench with basic precautions 
• Be sure you are trained in general mouse euthanasia and dissection before attempting the procedure 
• If you have many samples to process, it’s advisable to recruit help for the initial tissue rinsing and final cell straining

Prepare the following materials and reagents

• 20 mL strip buffer for each mouse/sample, warmed to 37C 
• Two labeled 50 mL conical tubes for each sample/mouse
• Two 70 um cell strainers for each sample/mouse
• Cold PBS (without Mg/Ca): in a bottle on ice, and in a petri dish for rinsing
• Cold DMEM media containing >5% serum and pen/strep antibiotics.
• Rocking platform and a 50 mL tube rack arranged to hold tubes horizontally 
• Waste bucket with a strainer at your work station throughout the procedure

Mouse dissection and preparation

• Do not forget to pre-warm strip buffer at 37C (20 mL per sample)
• Add ~5 mL cold wash media to one set of labeled 50 mL conical tubes and keep on ice
• Dissect gross intestinal tissues of interest (SI, ileum, colon, etc.) for your experiment
  • Optional: remove Peyer’s patches from small intestine with shallow snips of your scissors perpendicular to the gut tube before proceeding
• Open tissue longitudinally by sliding tissue onto one blade of your scissors and periodically cutting down
• Using forceps, vigorously rinse opened gut tissue a dish of cold PBS; place in 50 mL conical with media on ice
  • As needed, refresh PBS in dish
• Dissect all tissue samples on ice before proceeding

Epithelial strip

• Briefly vortex each sample in 50mL conical with media and, working one at a time, dump media+tissue into kitchen strainer set over waste bucket
• Using forceps, return tissue to the same tube; pour in ~20-30 mL cold PBS into tubes; work quickly, exact volumes are unimportant
• Briefly vortex each sample in PBS and dump PBS+tissue into strainer set over waste bucket
• Using forceps, return tissue to the same tube; pour in ~20 mL warm strip buffer; DO NOT return to ice
• Close tubes tightly and start a 20 min timer. Place tubes horizontally in racks on a rocking platform at 37C
• Set the rocking platform to vigorously but not frantically agitate the samples
• After 20 minutes, promptly place samples on ice and keep them there for the remainder of the procedure

Recover epithelial and epithelial-associated immune cells

• Working one at a time, vortex each sample vigorously for 5-10 seconds and pour through a 100 um cell strainer into a fresh tube
  • CRITICAL: do not omit vortexing. If you under-vortex at this phase, your cell recovery may be low.
  • Straining is best done with the destination tube in a rack on the bench
  • You will likely have to adjust the cell strainer to allow cells to freely flow into destination tube
  • If first strainer clogs, use a new strainer – pour whatever remains in initial strainer into the basin of the new one
  • Tissue may fall into the cell strainer. This is fine. Return the tissue to the initial tube for washing.
• Wash tissue with ~10 mL wash media to dilute and inactivate strip buffer and recover additional cells
  • Pour ~10 mL cold wash media into initial tube with tissue and vortex each sample vigorously for 5-10 seconds
  • Strain wash media as previously
  • Discard tissue and cell strainer, mix tubes briefly by inverting, and place on ice until all samples are collected
• Once all samples have been processed, centrifuge at 500RCF, 4C, 5 minutes to pellet
• Decant supernatant into waste bucket and resuspend cells in residual media
• Proceed with downstream applications. Add bleach to waste (10% final volume) and dispose of down drain after 10 min