Neutravidin resuspension:

Resuspended lyophilized 10 mg glass vial with 1 mL of ultrapure H2O (milliQ water) to make 10 ug/uL solution. Ensure that all neutravidin is dissolved, then dilute to 10 mL in 1X PBS. Make 1 mL aliquots and store at -20 deg C. Active aliquot can be kept at 4 deg C for weeks to months.

Anti-CD3 and anti-CD28 biotinylation:

Stock concentration of 2C11 (anti-CD3) from BioXcell: 2.8 mg/mL

Stock concentration of 37.51 (anti-CD28) from BioXcell: 2.0 mg/mL

1. Weigh 2.5 mg of EZ-Link Sulfo-NHS-SS-Biotin (Thermo part #21331) and resuspend in 250 uL of milli-q water to make 10 ug/uL solution. Biotin solution must be made fresh.
2. Make or find 1 M sodium bicarbonate solution, enough to add 10% of final volume
3. Mix freshly made biotin solution to antibody at a ratio of 8 uL per mg of antibody, then add 10% volume of 1 M sodium bicarbonate, example volumes:

360 uL of 2C11 stock solution  ~ 1 mg antibody,

+ 8 uL biotin

+ 36 uL bicarbonate

430 uL of 37.51 ~ 0.86 mg

+ 7 uL biotin

+ 43 uL bicarbonate

1. Tilt for 4 hrs at room temperature
2. Pipette into correct capacity Slide-a-lyzer G2 dialysis cassette, 2 kD molecular weight cutoff (3 mL capacity is part # P187718 from Fisher) according to cassette instructions
3. Place into 4 L large plastic beaker full of 4 liters of freshly made 1X PBS (diluted from 10X PBS) and leave overnight at 4 degrees
4. Aliquot and freeze
5. Note, concentration of antibody goes down by ~10%, for given concentrations indicated above 2C11 goes from stock concentration of 2.8mg/mL to 2.5 mg/mL after biotinylation, 37.51 goes from 2.0 mg/mL to 1.8 mg/mL         

Plate coating and T cell activation

1. Dilute neutravidin from 10 ug/uL stock to 5 ug/mL (1:200) in sterile PBS
2. Pipette 6 mL of diluted neutravidin in PBS into 10 cm Corning dishes (part # 430167) and leave in 37 d incubator for 3 hours while you harvest mouse T cells
3. Before plating, aspirate dilute neutravidin from Corning plates
4. Wash once with an equivalent amount of PBS and aspirate til dry immediately before plating T cells (don’t let plate sit dry for long)
5. Resuspend T cells in correct media, cytokines and antibodies and add biotinylated 2C11 to final concentration of 0.25 ug/mL and biotinylated 37.51 to final concentration of 1.0 ug/mL