Preparation and use of stimulation beads

Anti-CD19 Idiotype Antibody Bead Production and Use Protocol

1.     Borate Buffer (100 mL): 0.1M Borate solution

a.     Dissolve 0.618 mg of Boric acid in 95 mL diH₂O

b.     Adjust pH to 9.5 using NaOH (5N) by adding dropwise

c.     Adjust to 100 mL with diH₂O

d.     Filter through .22 µm sterile filter

e.     Store at 4°C

2.     Wash Buffer (1L):

a.     30 mL heat inactivated human AB serum

b.     4 mL 0.5 M EDTA

c.     10 mL 10% Sodium Azide

d.     956 mL PBS (w/o Ca²⁺ and Mg²⁺)

e.     Sterile Filter with .22 µm filter

f.      Store at 4°C

3.     Prep Antibody with Borate buffer (sterile): 

a.     1 mL buffer + antibody per 1 mL beads

b.     Add sufficient antibody to 1 mL of buffer to achieve 150 µg/mL antibody concentration

4.     Day 1 of prep Dynabeads™ M-450 Tosylactivated (sterile):

a.     Vortex Beads

b.     Pipette 1 mL Beads into 1.5 mL sterile screw-top microcentrifuge tube

c.     Place tube on magnet and remove supernatant

d.     Wash beads once with 1 mL borate buffer

e.     Plate tube on magnet and remove supernatant

f.      Resuspend beads in antibody/borate mix

g.     Place beads in a rotator at 37°C overnight

5.     Day 2:

a.     Place Bead on Magnet

b.     Discard supernatant

c.     Resuspend beads in 1 mL wash buffer

d.     Place beads on turner at 4°C for 10 minutes

e.     Repeat steps a-d 2X

f.      Once 3^rd wash has been completed, discard place beads on magnet

g.     Discard supernatant

h.     Resuspend beads in 1 mL wash buffer

i.      Place beads on turner at 4°C overnight

6.     Day 3:

a.     Place Bead on Magnet

b.     Discard supernatant

c.     Resuspend beads in 1 mL wash buffer

d.     Count beads at 1:100 dilution in PBS using coulter counter (can’t count at full concentration because it is too high for the coulter to get an accurate measurement)

e.     Record bead concentration

f.      Store beads at 4°C

7.     Day of use

a.     Pipet volume of beads needed for experiment into sterile screw-top tube

b.     Place beads on Magnet

c.     Discard supernatant

d.     Wash beads in 1 mL PBS

e.     Repeat b-d 2x

f.      Discard supernatant

g.     Resuspend beads in same media as used to culture cells

h.     Add beads to cells at 5:1 ratio (beads:cells) if performing signaling assay, or 3:1 ratio if performing an expansion assay

                                               i.     For signaling assays, the final volume should be adjusted to achieve a final concentration of 5e6 cells/mL and a plate with a final surface area of approximately 3.15e6 cells/cm²

                                             ii.     For expansion assays, the final volume should be adjusted to achieve a final concentration of 1e6 cells/mL and a plate with a final surface area of approximately 0.29e6 cells/ cm²

i.      For signaling assays:

                                               i.     Mix the cell-bead co-culture 

                                             ii.     Spin at no more than 300xg for 5 minutes to synchronize cell-bead contact

                                           iii.     Place in standard cell culture incubator at 37°C

j.      For expansion assays:

                                               i.     Mix the cell-bead co-culture

                                             ii.     Place in standard cell culture incubator at 37°C

Category