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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;amp;tool=pmcentrez&amp;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. 
#[[Preparation_of_Silica-Magnetic_Beads|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.

== Protocol ==

#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 [[Synchronised Phagocytosis|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.

 

== Recipes ==

===== Wash Buffer =====

*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 

===== Lysis Buffer =====

*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)

 

== References ==

<references />

Phagosome Isolation - Revision history

Admin: Created page with "This isolation is based on the protocol of Lonnbro ''et alhttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2453110&amp;amp;amp;amp;tool..."

Admin: Created page with "This isolation is based on the protocol of Lonnbro ''et alhttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2453110&amp;amp;tool..."