This isolation is based on the protocol of Lonnbro et al<ref name="Pubmed Central">http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2453110&amp;amp;amp;tool=pmcentrez&amp;amp;amp;rendertype=abstract</ref><ref name="Pubmed">http://www.ncbi.nlm.nih.gov/pubmed/18588680</ref>, and utilizes magnetic beads and gentle lysis to recover phagosomes.
Prior to Experiment:
- Grow cells to desired confluency - typically 60%-80% - using normal cell cluture methods.
- Following standard protocols prepare opsonized magnetic beads (e.g. IgG-coated, lipid-coated, etc).
- Prepare all required buffers, including wash & lysis buffers. Pre-cool and pre-warm all media, incubators, etc before begining procedure.
- Pre-cool nitrogen bomb in fridge at least 4 hours prior to use.
- Note: All steps should be conducted in HPMI-buffered media.
- Pre-suspend beads in the total volume of media required to cover the plate(s), cool to 10C.
- Wash cells 2X in 10C HPMI-buffered serum-containing media, then place in 10C fridge and allow to cool completely (~10 min). Use a large volume of media for these washes to ensure rapid cooling of cells.
- Once cells are cooled, replace media with bead-containing media (1ml for 5cm plates, 300ul for 35mm wells). Is possible use 100xG, 1min centrifugation to spin magnetic target particles onto phagocytes as described in synchronized phagocytosis. Otherwise, add an additional 5 min to the incubation (step 4)
- Incubate at 10C for 20 min to allow binding of particles, then was 2X in 37C serum-containing media to remove unbound particles and initiate phagocytosis. Large volumes (75% of max well volume) of warmed media should be used for this step to aid in rapid warming of the cells. Do not wash vigorously.
- Place in 37C incubator for the desired length of time, then wash 2X with ice-cold media and palce on-ice to cool.
- Add lysis buffer (~0.3ml per 35mm, ~1ml per 5cm plate, ~3ml for 10cm plate) and scrape cells into buffer. If using multiple wells per condition, transfer buffer from well-to-well instead of using additional buffer for each well.
- Pressurize lysate in pre-cooled nitrogen bomb to 300PSI. Let sit 10 min, then recover drop-wise into a parafilm-covered 1.5ml tube.
- Check a small sample on a slide for lysis; repeat step 9 if lysis is incomplete.
- Let sit on-ice for 10 min to allow DNAse to degrade DNA.
- On-ice, place tube in magnetic column and allow magnetic beads to accumulate on magent, approximately 10 min. If solution is still viscous add additional DNAse and retry recovery.
- Wash recovered beads in 1ml wash buffer. Recover with magnetic column; 5 min.
- Repeat step 11 an additional 1-2 times.
- After the last wash, carefully remove all of the supernatant being careful to not disturb the beads.
- Pellet beads by spinning tube, maximum speed, for 3 min in the minifuge.
- For immunoblotting or mass spectometry, solubilize phagosomes in lammelli's buffer with protease and phosphatase inhibitors and run on gel as per normal.
- For other experiments, treat isoalted phagosomes as required.
- 0.25M sucrose (856mg/10ml)
- 10mM HEPES (24mg/10ml)
- 3mM MgCl (3ul/10ml of a 1M stock)
- 1mM NaVO4 (100ul of 1M) - add immediately before use
- 1ml wash buffer
- 0.25mM PMSF (6.25ul of 200mM in ethanol)
- 200nM Okadaic acid (1ul of 1mM in DMSO)
- 10mM NaF (100ul of 500mM)
- Commercial protease and phosphatase inhibitors at the recommended concentration
1:50 dilution of DNAse I (10mg/ml stock)