Jun 18, 2019
Version 2
Phagocytosis Bead Conjugation V.2
- 1Dartmouth College, Ackerman Lab
- Ackerman Lab
Protocol Citation: Andrew Crowley 2019. Phagocytosis Bead Conjugation. protocols.io https://dx.doi.org/10.17504/protocols.io.4cygsxw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 18, 2019
Last Modified: June 18, 2019
Protocol Integer ID: 24696
Keywords: microsphere, bead, phagocytosis, Luminex, fluorescent
Abstract
Protocols for the amine coupling of fluorescent microspheres with antigen or capture reagent for opsonization and phagocytosis
Quantity as written: approx. 1 x 108 under typical yields (enough for 400 phagocytosis wells under standard protocol)
Materials
STEP MATERIALS
PBS-TBNTeknovaCatalog #P0210
PBS-TBNTeknovaCatalog #P0210
Protocol materials
PBS-TBNTeknovaCatalog #P0210
PBS-TBNTeknovaCatalog #P0210
PBS-TBNTeknovaCatalog #P0210
PBS-TBNTeknovaCatalog #P0210
Activation
Activation
Calculate the beads / mL from the percent solids as follows:
(6W x 1012) / (ρ x π x φ3)
W = grams solid per mL
ρ = density (g/mL)
φ = particle diameter (µm)
polystyrene density = 1.055 /mL
Resuspend beads by briefly vortexing; dispense 200 µL (contains appox. 7 billion beads)
Pellet by centrifugation 17000 x g 00:03:00 and remove supernatant by pipette
Resuspend in a total of 1 mL consisting of:
- sulfo-NHS 50 mg/mL made in 10 millimolar (mM) MES (pH 5.0) 100 µL
- EDC 50 mg/mL , made in 10 millimolar (mM) MES (pH 5.0) 100 µL
- 10 millimolar (mM) MES (pH 5.0) 800 µL
Incubate with end-over-end mixing (shielded from light) 00:20:00 Room temperature
Coupling
Coupling
Wash by pelleting beads and replacing supernatant with 10 millimolar (mM) MES (pH 5.0) 1 mL
17000 x g 00:02:00
Formulate protein for coupling @ approx. 25 µg/mL in 10 millimolar (mM) sodium acetate (pH 5.0) 7.5 mL
Note
n.b.
The large volume used for the step is based on observations that sufficiently high bead concentrations during this step may lead to crosslinking between microspheres (anecdotal)
Incubate with end-over-end mixing (shielded from light) 03:00:00 Room temperature
Blocking + Washes
Blocking + Washes
Collect the full volume in the base of the reaction tube by centrifugation 250 x g 00:01:00
Split the 7.5 mL volume into multiple fractions in microcentrifuge tubes (2 mL ) in order to centrifuge at a sufficiently high speed 17000 x g 00:03:00
Remove supernatant from each fraction by pipette
Resuspend one fraction in PBSF (ie. 1x PBS + 0.1 Mass / % volume BSA) 1 mL
Transfer the entire contents of the first fraction to the second and resuspend; continue through the fractions until as many beads as possible have been collected in a single microcentrifuge tube
Pellet beads via centrifuge 17000 x g 00:02:00
go to step #11 for a total of 3 washes
Resuspend in PBSF 500 µL
Will the beads be used for an assay on the same day as production?
No16 steps
Block via 16:00:00 (ie. overnight) 4 °C
Counting + Qualification
Counting + Qualification
Make serial dilutions of 1:100, 1:1000, and 1:10,000
Measure concentration by flow cytometry using the fluorescent dye of the microsphere as the trigger condition
Expected result
Typical yield is approx. 1 - 2 x 108 beads, or around 25% of the original amount
Equipment
MACS Quant
NAME
flow cytometer
TYPE
Miltenyi
BRAND
130-096-343
SKU
In a non-binding, 96-well plate, formulate serial dilutions of positive and negative control antibodies beginning at 5 µg/mL and diluted by 1:3 over an approx. 4 point series
Add 100,000 beads to each well
Incubate with shaking (shielded from light) 01:00:00 Room temperature
Pellet by centrifugation 3500 x g 00:05:00 and remove supernatant by decanting
Resuspend beads in PBST (ie. 1x PBS + 0.05 % volume Tween20) 200 µL
go to step #24 for a total of 2 washes
Resuspend beads in fluorescent secondary 100 µL 0.65 µg/mL
Note
n.b.
Make sure that the fluorescent molecule chosen for the secondary does not have spectral overlap with the dye in the microspheres themselves
Incubate with shaking (shielded from light) 00:30:00 Room temperature
Pellet by centrifugation 3500 x g 00:05:00 and remove supernatant by decanting
Resuspend beads in PBST (ie. 1x PBS + 0.05 % volume Tween20) 200 µL
Pellet by centrifugation 3500 x g 00:05:00 and remove supernatant by decanting
Resuspend beads in 1x PBS 100 µL
Measure fluorescent intensity by flow cytometry
Equipment
MACS Quant
NAME
flow cytometer
TYPE
Miltenyi
BRAND
130-096-343
SKU