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Opsonization allows phagocytes to efficiently recognize particles, by coating them with opsonins such as IgG isotype antibodies. This page contains the protocols for opsonising particles, coverlslips, and related information:
 
== Polystyrene Beads: ==

Important Note: Polystyrine beads are ordered from [http://bangslabs.com/ Bangs Laboratories].  These beads come in several sizes and compositions.  For best results, always use beads '''with''' 2% DVB; otherwise opsonizing proteins will not bind the beads.  Size is also important; for most studies beads of 5μm are used, but sizes from 1-8μm may be used.  Please note that the signalling requirements for smaller beads may be different than for larger beads.

#Mix:
#*100μL PBS
#*10μL beads (vortex well before using)
#*10μL human or mouse IgG (stock is 50mg/mL)

#<li value="2">Incubate 30-60min, shaking at 37oC, or 4oC overnight<li>
#Wash in 1mL PBS and re-suspend in 100μL PBS
#For a spaceship (35mm well) add 10-20μL of this suspension for minimal cups; add 4X for lots of cups.

 

== Sheep Red Blood Cells (sRBCs): ==

#Lightly vortex the sRBCs to get a homogenous solution
#Draw 200μL from the bottle (kept in the fridge) with a 1mL syringe
#Spin 10 sec in the microfuge, aspirate supernatant and resuspend by pipetting gently in 1mL PBS
#Spin and aspirate as before
#Re-suspend in 200μL PBS with 4μL (1/50) Rabbit anti-sheep IgG (same fridge)
#Incubate 1h shaking (level 9) at 37°C
#Wash 3x PBS, resuspend in 200μL PBS
#Add 5μL to a single well of a 12 well (scale as necessary)

 

== Aggrigated IgG: ==

#Mix:
#*100μL PBS
#*20μL IgG (50mg/ml stock)
#<li value='2'>Incubate 30' without shaking at 62°C <li>
#Spin down @ 16000g for 10'
#Add 1:50 of supernatant

Reference: PMID16455948<ref>http://www.ncbi.nlm.nih.gov/pubmed?term=16455948</ref> 

 
== Coverslips (IgG) ==
Typically used for frustrated phagocytosis
#Wash coverslips in 70% EtOH
#Wash 2x with PBS
#Add 500μL or 800μL (for 18 or 25mm coverslips respectively) 1mg/mL huIgG in PBS
#Shake (orbital) 60' at RT
#Rinse 2x PBS and store @ 4°C under PBS until required

===== Alternative method 1 =====
Instead of using IgG bound to glass (which can result in IgG binding in random orientations) you can first coat the coverslip as described in PMID9382884<ref>http://www.ncbi.nlm.nih.gov/pubmed?term=9382884</ref>. Briefly:
#Acid-wash coverslips are coated with poly-L-lysine (100 μg/ml) and treated with 2.5% glutaraldehyde for 15 min.
#Coverslips are then washed and coated with BSA (1 mg/ml) for 30 min and then blocked with 0.1 M glycine for 2 h.
#To create immobilized BSA/anti-BSA immune complexes, BSA-coated coverslips are incubated with 40 μg of rabbit anti-BSA IgG in PBS for 1 h.

===== Alternative method 2 =====
#Add 0.1% BSA (0.1g/100mL) in PBS to coverslips for 1-2h
#Wash off and treat with a mouse α BSA antibody (monoclonal). 1:10 highly activates cells (very fast spreading), 1:100 for slightly slower spreading.
#Wash 2x PBS
 
== References: ==
<references />

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