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:
Important Note: Polystyrine beads are ordered from 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.
- 100μL PBS
- 10μL beads (vortex well before using)
- 10μL human or mouse IgG (stock is 50mg/mL)
- Incubate 30-60min, shaking at 37oC, or 4oC overnight
- 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)
- 100μL PBS
- 20μL IgG (50mg/ml stock)
- Incubate 30' without shaking at 62°C
- Spin down @ 16000g for 10'
- Add 1:50 of supernatant
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