TITLE: Phagocytosis Bead Conjugation
AUTHORS: Andrew Crowley

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocols for the amine coupling of fluorescent microspheres with antigen or capture reagent for opsonization and phagocytosis</div><div class = "text-block"><span>Quantity as written: approx. 1 x 10</span><span style = "vertical-align:super;">8</span><span> under typical yields (enough for 400 phagocytosis wells under standard protocol)</span></div></div>

[STEPS]
?. [Activation]
Resuspend beads by briefly vortexing; dispense
[(contains appox. 7 billion beads)]
?. [Activation]
Pellet by centrifugation  and remove supernatant by pipette
?. [Activation]
Resuspend in a total of  consisting of:    - sulfo-NHS  made in  MES (pH 5.0)     - EDC , made in  MES (pH 5.0)     -  MES (pH 5.0)
1 ml
100 µl
100 µl
800 µl
?. [Activation]
Incubate with end-over-end mixing (shielded from light)
0 Room temperature
?. [Coupling]
Wash by pelleting beads and replacing supernatant with  MES (pH 5.0)
1 ml
?. [Coupling]
Formulate protein for coupling @ approx.  in  sodium acetate (pH 5.0)
7.5 ml
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)
?. [Coupling]
Incubate with end-over-end mixing (shielded from light)
0 Room temperature
?. [Blocking + Washes]
Split the  volume into multiple fractions in microcentrifuge tubes () in order to centrifuge at a sufficiently high speed
7.5 ml
2 ml
?. [Blocking + Washes]
for a total of 3 washes
?. [Blocking + Washes]
Resuspend in PBSF
500 µl
?. [Blocking + Washes]
Collect the full volume in the base of the reaction tube by centrifugation
?. [Blocking + Washes]
Remove supernatant from each fraction by pipette
?. [Blocking + Washes]
Resuspend one fraction in PBSF (ie. 1x PBS +  BSA)
1 ml
?. [Blocking + Washes]
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
?. [Blocking + Washes]
Pellet beads via centrifuge
?. [Blocking + Washes]
Block via
[(ie. overnight)]
4 °C
?. [Counting + Qualification]
Make serial dilutions of 1:100, 1:1000, and 1:10,000
?. [Counting + Qualification]
Measure concentration by flow cytometry using the fluorescent dye of the microsphere as the trigger condition
Typical yield is approx. 1 - 2 x 108 beads, or around 25% of the original amount
?. [Counting + Qualification]
In a non-binding, 96-well plate, formulate serial dilutions of positive and negative control antibodies beginning at   and diluted by 1:3 over an approx. 4 point series
?. [Counting + Qualification]
Add 100,000 beads to each well
?. [Counting + Qualification]
Incubate with shaking (shielded from light)
0 Room temperature
?. [Counting + Qualification]
Pellet by centrifugation  and remove supernatant by decanting
?. [Counting + Qualification]
Resuspend beads in fluorescent secondary
100 µl
n.b.Make sure that the fluorescent molecule chosen for the secondary does not have spectral overlap with the dye in the microspheres themselves
?. [Counting + Qualification]
Resuspend beads in PBST (ie. 1x PBS +  Tween20)
200 µl
?. [Counting + Qualification]
for a total of 2 washes
?. [Counting + Qualification]
Pellet by centrifugation  and remove supernatant by decanting
?. [Counting + Qualification]
Resuspend beads in PBST (ie. 1x PBS +  Tween20)
200 µl
?. [Counting + Qualification]
Incubate with shaking (shielded from light)
0 Room temperature
?. [Counting + Qualification]
Pellet by centrifugation  and remove supernatant by decanting
?. [Counting + Qualification]
Resuspend beads in 1x PBS
100 µl
?. [Counting + Qualification]
Measure fluorescent intensity by flow cytometry
?. [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
?. [Activation]
Collect beads by pulse centrifugation  and place in magnetic microcentrifuge rack (shielded from light)
n.b.It can be helpful to partially open the tubes before placing them in the magnetic rack so that they can be opened all the way after incubation without disturbing the beads more than necessary
?. [Coupling]
Formulate protein for coupling @ approx.  in 1x PBS
250 µl
?. [Activation]
Wash by removing  of supernatant and replacing with  of 1x PBS; pulse vortex to mix and resuspend beads
90 µl
100 µl
This introduces a 10 µL excess that will protect against bead aspiration throughout the remainder of the protocol where equal volumes will be removed and added
?. [Activation]
Incubate with end-over-end mixing (shielded from light)
0 Room temperature
?. [Activation]
Remove supernatant and resuspend in a total of  consisting of:    - sulfo-NHS  made in  MES (pH 5.0)     - EDC , made in  MES (pH 5.0)     -  MES (pH 5.0)
90 µl
5 µl
5 µl
80 µl
?. [Washing + Blocking]
to separate beads from the supernatant
?. [Activation]
to separate the beads from the supernatant
?. [Coupling]
to separate the beads from the supernatant
?. [Coupling]
Remove supernatant and resuspend in protein of interest ()
250 µl
?. [Coupling]
Incubate with end-over-end mixing (shielded from light)
0 Room temperature
?. [Washing + Blocking]
Remove supernatant and replace with PBS-TBN
400 µl
n.b.Whenever volume of supernatant exceeds 200 µL, use a P200 pipette multiple times - the opening of a P1000 will aspirate too many beads
?. [Washing + Blocking]
for a total of 3 washes
?. [Washing + Blocking]
Remove supernatant and replace with PBS-TBN
250 µl
?. [Counting + Qualification]
?. [Washing + Blocking]
Block via
[(ie. overnight)]
4 °C
?. [Washing + Blocking]
Will the beads be used for an assay on the same day as production?
?. [Washing + Blocking]
Block via
0 Room temperature
?. [Blocking + Washes]
Will the beads be used for an assay on the same day as production?
?. [Blocking + Washes]
Block via
0 Room temperature
?. [Counting + Qualification]
Make serial dilutions of 1:100, 1:1000, and 1:10,000
?. [Counting + Qualification]
Measure concentration by flow cytometry using the fluorescent dye of the microsphere as the trigger condition
Typical yield is approx. 1 - 2 x 108 beads, or around 25% of the original amount
?. [Counting + Qualification]
In a non-binding, 96-well plate, formulate serial dilutions of positive and negative control antibodies beginning at   and diluted by 1:3 over an approx. 4 point series
?. [Counting + Qualification]
Add 100,000 beads to each well
?. [Counting + Qualification]
Incubate with shaking (shielded from light)
0 Room temperature
?. [Counting + Qualification]
Pellet by centrifugation  and remove supernatant by decanting
?. [Counting + Qualification]
Resuspend beads in PBST (ie. 1x PBS +  Tween20)
200 µl
?. [Counting + Qualification]
for a total of 2 washes
?. [Counting + Qualification]
Resuspend beads in fluorescent secondary
100 µl
n.b.Make sure that the fluorescent molecule chosen for the secondary does not have spectral overlap with the dye in the microspheres themselves
?. [Counting + Qualification]
Incubate with shaking (shielded from light)
0 Room temperature
?. [Counting + Qualification]
Pellet by centrifugation  and remove supernatant by decanting
?. [Counting + Qualification]
Resuspend beads in PBST (ie. 1x PBS +  Tween20)
200 µl
?. [Counting + Qualification]
Pellet by centrifugation  and remove supernatant by decanting
?. [Counting + Qualification]
Resuspend beads in 1x PBS
100 µl
?. [Counting + Qualification]
Measure fluorescent intensity by flow cytometry