DNA reagents (thiol-DNA and compDNA)
Calculate volume needed to resuspend DNA to 500 µM. Resuspend using 10 mM HEPES, pH 7.0. Allow for 30 minutes to resuspend, vertexing occasionally. Store at -20°C.
5x Particle fabrication buffer, 50 mM sodium citrate, 1.5 M sodium chloride, 10 mM magnesium chloride, pH 3.0
Weigh out 735 mg of sodium citrate, 4.38 g of sodium chloride, and 47.61 mg of magnesium chloride. Add into a container with 50 mL of deionized water and mix. Measure pH using a pH meter and adjust to pH 3.0 using concentrated hydrochloric acid. Transfer to conical tubes and store at room temperature.
2x DNA hybridization buffer, 600 mM sodium chloride, 2 mM magnesium chloride, 0.02% Tween 20, pH 7.0
Weigh out 1.75 g of sodium chloride and 9.52 mg of magnesium chloride. Add into a container with 50 mL deionized water. Add 10 µL of Tween 20, using a dilution in water if the stock is too viscous to accurately measure. Thoroughly mix and measure pH using a pH meter and adjust to pH 7.0. Store at room temperature.
5x Protein G binding buffer, 100 mM sodium phosphate, 0.75 M sodium chloride, pH 7.0
Weigh out 3.0 g of sodium phosphate monobasic and 10.88 g sodium chloride. Add into a container with 250 mL of deionized water. Thoroughly mix and measure the pH using a pH meter and adjust to pH 7.0. Store at room temperature.
Protein G acidic elution buffer, 0.1 M glycine, pH 2.7
Weigh out 375.35 mg of glycine and add to a container with 50 mL deionized water. Thoroughly mix and measure the pH using a pH meter and adjust to pH 7.0 using concentrated hydrochloric acid. Store at room temperature.
Protein G basic elution buffer, 0.1 M glycine, pH 10.0
Weigh out 375.35 mg of glycine and add to a container with 50 mL deionized water. Thoroughly mix and measure the pH using a pH meter and adjust to pH 7.0 using concentrated sodium hydroxide. Store at room temperature.
10x Protein G acidic neutralization buffer, 1 M Tris-HCl, pH 8.5
Measure out 50 mL of 1 M Tris-HCl in a secondary container. Measure the pH using a pH meter and adjust to pH 8.5 using concentrated sodium hydroxide. Store at room temperature.
10x Protein G basic neutralization buffer, 1 M Tris-HCl, pH 6.5
Measure out 50 mL of 1 M Tris-HCl in a secondary container. Measure the pH using a pH meter and adjust to pH 6.5 using concentrated hydrochloric acid. Store at room temperature.
PBS-FBS wash buffer, 1x Ca2+/Mg2+ free PBS, 3% FBS (v/v), 1 mM EDTA
In a sterile BSC, combine 15 mL of heat-inactivated FBS, 484 mL of 1x PBS, and 1 mL of 0.5 M EDTA in a container and mix. Mix and sterile-filter the solution using a 0.22 µM filter. Store at 4°C.
T cell medium
In a sterile BSC, combine 435 mL of RPMI 1640 + Glutamax, 50 mL of heat-inactivate FBS, 5 mL of 1 M HEPES, 5 mL of 100 mM sodium pyruvate, and 5 mL of combined Penicillin (1 x 104 U/mL) + Streptomycin (10 mg/mL). Mix and sterile-filter the solution using a 0.22 µM filter and store at 4°C. Prior to experimental use, aliquot 50mL of media into a separate container and add 25 µL of hIL2 (2 x 105 U/mL stock) to a final concentration of 100 U/mL. Media containing hIL2 (complete T cell media) can be used for T cell culturing and should be used within 1 week and stored at 4°C.
Freezing medium, 10% DMSO in FBS (v/v)
In a sterile BSC, combine 22.5 mL of heat-inactivated FBS and 2.5 mL of DMSO. Sterile-filter using a 0.22 µM filter and store at 4°C.
PLGA-PEG-DNA conjugate synthesis
Critical: The following describes the synthesis of 500 nmol PLGA-PEG-DNA using commercially synthesized PLGA-PEG-Mal and thiol-DNA precursors. See Table 1 for validated DNA-sequence options. Repeat the procedure for each desired oligo sequence.
1. Use a micropipette to transfer 500 nmol of thiol-DNA into a 1.5 mL microcentrifuge tube (DNA-tube). Add 100 µL of 500 mM TCEP (100x molar excess to thiol-DNA) to reduce any inter-strand disulfide bonds and incubate for 1.5 hours at 37°C.
2. Prepare a Glen size-exclusion column that is appropriately sized for DNA-tube volume and buffer-exchange the TCEP-reduced thiol-DNA per the manufacturer’s instructions, collecting the DNA-containing flow-through. Use 10 mM EDTA in 10 mM Tris-HCl (1x TE, pH 7.5) for buffer exchange washes.
Critical Step: The exchange buffer should not contain any chemical groups which react with the selected conjugation chemistry. EDTA prevents disulfide reformation following reduction.
3. For DNA precipitation, aliquot the thiol-DNA into 1.5 mL tubes (precipitation tubes) with ~400 µL per tube. To each 400 µL tube, add 50 µL of 3 M sodium acetate (pH 5.0) and 1.3 mL of ethanol (200 proof); thoroughly mix and vortex after each addition. Cool tubes at -20°C for 30 minutes.
4. Centrifuge the precipitation tubes at 18000 g for 10 minutes at 4°C. Remove the supernatant and either air-dry or use a pressurized air-line to further dry the DNA-pellet.
5. Resuspend the DNA-pellet in one precipitation tube with 200 µL of TE. Combine this volume into another precipitation tube and repeat until all tubes are resuspended in a total of 200 µL (targeting ~2.5 mM DNA if DNA loss was minimal during preceding steps).
Critical Step: DNA should be resuspended in less than 200 µL to be compatible with the optimized reaction conditions later. Adjust this volume appropriately and reoptimize if needed.
6. Measure the absorbance at 260 nm (A260) of a diluted sample of DNA using Nanodrop. Reference Table 1 for the relevant extinction coefficients and calculate the stock concentration using Beer’s law:
a. Stock Concentration= [(Dilution Factor) x (Absorbance)] / [(extinction coefficient) x (path length)], where Nanodrop path length is 1 cm and the extinction coefficients used here are in the units of M-1 × cm-1.
7. Create a reaction-template in Excel to facilitate reagent calculations for synthesizing the PLGA-PEG-DNA. The reaction occurs in a 90% organic (DMF) and 10% aqueous (TE). The aqueous phase constitutes the 500 nmol of DNA and additional TE buffer needed to reach 10% aqueous final volume. The organic phase contains enough dissolved PLGA-PEG-Mal to match the desired PLGA-PEG-Mal : DNA molar ratio, the basic catalyst triethylamine to yield 1% of the total reaction volume, and additional DMF to reach 90% organic phase in the total volume. Refer to Table 2 for the necessary equations and constants for constructing the template.
Critical Step: The PLGA-PEG-Mal : DNA ratio should be optimized for each new polymer lot. Use PLGA-PEG-Mal molecular number average instead of weight average due to the distribution of different polymer chain lengths. The number average here is specific to our PLGA-PEG-Mal lot.
8. Allow the PLGA-PEG-Mal container to warm to room temperature before opening. Weigh the calculated amount of PLGA-PEG-Mal and add DMF to achieve a 30 mg/mL solution.
Critical Step: Allowing the container to warm to room temperature before opening to avoid water condensation, which can hydrolyze the functional group.
9. To a 15 mL tube, add the solutions in the following order, referring to the volumes in the reaction template: 1) DNA solution, 2) extra TE buffer, 3) triethylamine, 4) Extra DMF, 5) PLGA-PEG-Mal DMF solution. Vortex to mix, wrap the top of the tube with parafilm, and shake overnight using an orbital shaker at room temperature.
10. Use nitrogen or other inert gas line to back-fill the stock container of PLGA-PEG-Mal. Wrap the container with parafilm before putting back into -20°C storage.
11. The next day, briefly vortex the PLGA-PEG-DNA reaction tube and aliquot into 1.5 mL tubes with ~500 µL into each tube. Since ratiometric particles may be desired, it is recommended to premix PLGA-PEG-DNA bearing different sequences at a specified ratio before drying, ensuring that 100 nmol of total PLGA-PEG-DNA is aliquoted per tube. Dry the PLGA-PEG-DNA aliquots in a vacuum centrifuge at 70°C for 2-3 hours. Once dried, store at -20°C.
Critical Step: The downstream fabrication protocol uses 100 nmol of PLGA-PEG-DNA, thus aliquoting 500 µL equates to a theoretical 100 nmol of PLGA-PEG-DNA (assuming 200 µM was the target PLGA-PEG-Mal reaction concentration). Premixing the different PLGA-PEG-DNA sequences prior to drying ensures more precise control over the mixture ratio, whereas later the volumes may be difficult to control due to solvent evaporation.
Pause Point: Dried PLGA-PEG-DNAs are stable for over a year. PLGA-PEG-DNA can be stable if dissolved in organic solvent, although any aqueous solutions should be avoided as this will lead to hydrolysis of either the PLGA ester linkages or the thiol-maleimide bond.
12. Urea-PAGE gel electrophoresis is used to verify PLGA-PEG-DNA conjugation. Prepare ~20 µL of a 0.2 µM solution of PLGA-PEG-DNA (diluted in 1x TE) and dilute to 0.1 µM using 20 µL of 2x Urea-PAGE loading buffer. Similarly, make a 0.1 µM dilution of pure-DNA (in loading buffer) used for the reactions. Heat the sample for 3 minutes at 70°C.
13. During heating, prepare a Urea-PAGE gel by loading a vertical gel-chamber with 1x TBE buffer and pre-running the gel for 10 minutes at 120 V. Use a syringe or pipette to clean the melted gels in each lane using TBE buffer within the chamber.
14. Load 1 pmol (~10 µL) of 0.1 µM sample in triplicate alongside 1 pmol of control pure-DNA lanes. Run the gel for 1.5 hours at 120 V.
15. Prepare a 25 mL of 1x Sybr Gold (10000x dilution) in 1x TBE. Dispense into a wide disposable glass dish, cover the dish with the lid, and protect from light. After the gel has finished running, release the gel from the cast and transfer to the 1x Sybr Gold solution. Place onto an orbital shaker at room temperature for 5-10 minutes protected from light.
16. Rinse the stained gel with 1x TBE and transfer into a new glass dish containing buffer to prevent gel dehydration. Image the gel using a gel-doc reader or laser-scanner.
17. Import the gel image into ImageJ. After adjusting brightness and contrast, measure the intensity of the top PLGA-PEG-DNA band and the lower, unreacted DNA. Calculate the efficiency of the reaction using the below equation and record to track batch variation:
a. (Intensity PLGA-PEG-DNA) / (Intensity PLGA-PEG-DNA + Intensity DNA)
PLGA particle fabrication
Critical: This procedure describes the fabrication of 1.5 µm diameter particles bearing a maximally dense surface DNA scaffold at 1:1, R:G DNA-sequence ratios (see Table 1 for sequence information), where R and G are different DNA sequences. This procedure assumes that 100 nmol of PLGA-PEG-DNA was dried in Step 11 with a 1:1 mixture of DNA -G and -R sequences (PLGA-PEG-G and PLGA-PEG-R, respectively). 100 nmol of PLGA-PEG-DNA generates ~100 OD550 in 400 µL volume (40 OD550 in 1 mL) or approximately 2 x 109 particles. For fabricating particles of other target diameters, refer to Table 3.
18. Weigh 50 mg of unmodified PLGA 50:50 (38-54 kDa, PLGA) into a 15 mL tube (fabrication tube). Use a glass pipette to add 400 µL of ethyl acetate (EtOAc) into the tube. Wrap the tube with parafilm and place vertically on a shaker table overnight to dissolve.
Critical step: Keep EtOAc containing tubes open for as little time as possible to minimize evaporation—this will reduce the size variability between batches. Do not hold tubes near the liquid as this may contribute to heating.
19. The next day, place stock tubes of EtOAc, water, and fabrication tube on ice to reduce evaporation when opened. Resuspend the 1:1 R:G PLGA-PEG-DNA tube from Step 11 with 100 µL of water and 100 µL of EtOAc. Reuse this pipette tip whenever transferring PLGA-PEG-DNA for a given sequence ratio (switch if using a different sequence ratio).
20. Place the PLGA-PEG-DNA tube into the bath sonicator for 10 minutes or until fully resuspended.
21. Transfer the PLGA-PEG-DNA into the 15 mL fabrication tube in 100 µL increments to reduce material loss inside the pipette tip.
22. To wash the PLGA-PEG-DNA tube, add 300 µL of water and 100 µL of 5x particle fabrication buffer (see “Reagent Setup”). Using the saved PLGA-PEG-DNA pipette tip, transfer this solution into the fabrication tube. If the pipette tip gets clogged, briefly pipette the EtOAc fraction within the fabrication tube to dissolve the clog. Sonicate the fabrication tube and vortex until mixed. Place the fabrication tube on ice.
23. Place a magnetic stir plate with a 250 mL beaker and a stir magnet into a fume-hood. This will be needed after probe sonication after Step 27.
24. Prepare a 50 mL conical tube partially filled with ice to act as a secondary container for the fabrication tube during probe sonication. Set up a vortexer, 0.2% PVA (w/v in water), and separate ice container near the probe sonicator.
25. For sonication setup, clean the sonication microtip probe using 70% ethanol (v/v in water) and allow to dry. Ensure that the sonication program is set to the recommended settings (Box 1). Sonication will need to pause halfway through, so if your sonicator does not allow for this function then adjust the number of cycles accordingly.
26. Vortex the reaction tube and place into the 50 mL secondary ice container. Position the sonication probe into the fabrication tube solution, avoiding the tube walls. Initiate the sonication program, moving the microtip throughout the solution to ensure a more homogenous sonication. After two cycles, pause sonication and vortex the reaction tube before finishing the remaining cycles.
27. Immediately after sonication add 9 mL of 0.2% PVA (w/v) into the fabrication tube, invert to mix, then vortex. Dispense the contents of fabrication tube into the 250 mL beaker from Step 23 and turn on the magnetic stirrer for ~2.5 hours without any heating.
Critical Step: This step will evaporate the EtOAc residue. For larger volumes, use a rotary evaporator.
28. After 2.5 hours, place a 40 µm filter onto a 50 mL conical tube and pour the particle solution through the filter. Use a micropipette to transfer any remaining solution. Centrifuge the particle tubes at 225g for 10 minutes.
29. Discard the supernatant and resuspend in 2 mL of TE containing 0.1% Tween 20 (v/v, TE-Tween) using a micropipette. Distribute the 2 mL into smaller microcentrifuge tubes and centrifuge at 6000 g for 5 minutes.
30. Resuspend each tube in 200 µL of TE-Tween. Spin again at 6000 g for 5 minutes, resuspending again in 200 µL TE-Tween. During the final resuspension, combine all tubes into a single tube with a total volume of ~400 µL TE-Tween. Prepare a small sample for Nanodrop quantification. Measure the OD550 and calculate the stock concentration by multiplying by the dilution factor.
Critical Step: Microparticles settle quickly, creating a concentration gradient and a particle pellet over time. Whenever handling microparticles, ensure the tubes are sufficiently resuspended.
Critical Step: This reaction scale typically generates upwards of 100 OD550 of microparticles in 400 µL. Particle absorbance is linear between 0.2 and 1.0 at OD550, so the dilution fold would need to be adjusted accordingly.
31. Set aside a small sample of particles to generate ~20 µL at 5-10 OD. Save this sample for imaging and size-quantification later.
32. To each ~400 µL tube of particles, add 100 µL of 5% PVA (w/v) and mix.
33. In a secondary container, prepare a small volume of liquid nitrogen. Flash-freeze the tubes by submerging in liquid nitrogen below the cap-level using a tube holder (e.g. long-forceps). Place the frozen tubes into a lyophilization chamber for 24 hours with the tube-caps open.
Pause point: Lyophilized particles can be stored for up to two years at -20°C. Particles stored after two years should be reassessed for DNA-scaffold density (see “Particle surface DNA loading analysis”)
Particle surface DNA loading analysis
Critical: This protocol describes the quantification of particle scaffold DNA density and relative ratio of DNA sequences via the detection of hybridized, fluorescently labeled compDNA (5’ end label). The procedure assumes particles are taken from lyophilized stock.
34. Remove the lyophilized particles from Step 33 onto a disposable weigh-boat. Use a razor blade to cut a small fraction of the particle for OD550 measurement. Target a concentration of 20 OD550 in 100 µL and readjust later after OD550 quantification is made.
Critical Step: 20 OD550 in 100 µL was chosen to ensure that the particle signal will be above the signal detection limit for our spectrophotometer. Additionally, if users are not careful during pipetting steps there could be substantial particle loss, which is mitigated by increasing the initial particle quantity.
35. Resuspend particle sample in 500 µL of water for 5 minutes. Following this, centrifuge the particles at 6000 g for 5 minutes.
Critical Step: Since the particles were lyophilized in a non-volatile buffer, the buffer salts are still contained in the pellet. Water should be used to resuspend to prevent high concentrations of buffer salts.
36. Remove the supernatant and wash with 500 µL of TE-Tween. Repeat spinning and washing one more time with the final resuspension in 100 µL of TE-Tween. Make a sample dilution in a separate tube use the OD550 to calculate the stock tube concentration. If there is less than 20 OD550 in 100 µL, repeat Steps 37-38 to resuspend a newly cut portion of the particle as describe previously and add to the existing particle volume after sufficient wash steps. Repeat OD550 quantification.
37. Calculate the hybridization component volumes according to Table 4, assuming 20 OD550 of particle in 100 µL total hybridization volume.
Critical Step: The total loading capacity of compDNA of high scaffold density 1-2 µm diameter particles approaches 75-150 nM of compDNA per OD550 depending on batch-to-batch variation. For the 1:1 R:G particle here, both compR and compG DNAs will maximally load between 37.5-75 nM/OD550, respectively. CompDNA should be loaded at 3x maximal theoretical loading capacity (~225 nM/OD550 for each compDNA on the 1:1 particle) to ensure surface saturation regardless of particle batch variability.
Critical Step: The hybridization buffer will constitute half of the total volume. The remaining half will be used for particle volume and compDNA. If 100 µL has not been reached, calculate the volume for TE-Tween to fill the remainder. The total hybridization volume may exceed the target volume depending on the concentration of reagents, so extra TE-Tween may not be required (seen as a negative or zero value for the extra TE-Tween calculation).
38. Transfer a quantity of particles into a microcentrifuge tube such that, once diluted, will be at 20 OD550 in 100 µL (hybridization tube). If the volume of particles needed in Step 37 exceeds 50 µL then centrifuge particles and remove supernatant until 50 µL of volume remains.
39. To the hybridization tube, add 50 µL 2x hybridization buffer, compDNAs, and extra TE-Tween (if needed). Mix the solution using a micropipette followed by bath sonication for 15 seconds to ensure particle dispersion. Incubate particles on a shaker for 30 minutes at 37°C.
Critical Step: Particle hybridization is achieved in less than 2 minutes, although to ensure surface saturation we hybridize for 30 minutes. During this time, settling occurs at high particle concentrations. If this is substantial, vortex the particles halfway through their incubation period.
40. During particle incubation, generate fluorescent-compDNA standard curves in a black-walled microwell plate. For each fluorescent compDNA, start with a 200 µL of a 2 µM DNA concentration in 9% DMSO in PBS (v/v, PBS-DMSO). Remove 100 µL to perform 2-fold serial dilutions until reaching the limit of detection for the plate spectrophotometer, leaving 100 µL per well. Separately, make blank-wells containing 100 µL PBS-DMSO for background subtraction. Cover the well-plate top and set-aside to protect from light.
Critical Step: Particles will be loaded onto the plate in PBS-DMSO, so the ladder should be made in the same buffer.
41. After hybridization, add 400 µL of TE-Tween and centrifuge at 6000 g for 5 minutes at 4°C. Remove supernatant and wash twice more. Use 120 µL TE-Tween for the final resuspension.
Critical Step: After particles have been hybridized, all centrifugation steps should occur at 4°C to minimize dehybridization of loaded cargos.
Critical Step: It is important to remove a majority of the supernatant to prevent background signal. It is additionally important to not disturb the pellet during any steps, as this will reduce the total signal detected during later steps.
42. Add 50 µL of hybridized particles (particle replicate tubes) into two separate centrifuge tubes—these will be used for repeated measures. With the remaining 20 µL volume, dilute a small volume for OD550 calculation to determine the concentration in the particle replicate tubes. This value will be needed to calculate the final DNA nM/OD550.
43. Centrifuge replicate particle tubes at 6000 g for 5 minutes and remove 45 µL of supernatant from each. Add 45 µL of DMSO to each particle tube to dissolve particles.
Critical Step: 5 µL of wash buffer should be remaining after supernatant removal to reduce particle loss. If previous wash steps were not thorough, the 5 µL of remaining supernatant could include background DNA signal. The 45 µL of removed supernatant can be saved and measured to determine the background fluorescence contribution.
44. For replicate measurements, add 90 µL of PBS into the microwell plate from Step 40 and 10 µL of dissolved particles. Resuspend all wells thoroughly and do not generate bubbles.
45. Read fluorescence of the microplate on a microplate spectrophotometer in top-down mode with settings in accordance with the respective fluorophores used.
Critical Step: Filters should be carefully selected to minimize signal crossover between fluorophores. Other settings, such as channel voltage, should be optimized for each machine.
46. For fluorescence analysis, average the blank PBS-DMSO wells and subtract from all wells. Create a linear best-fit curve for the fluorescent ladder lanes.
Critical Step: Since the ladder fluorescent signal could be widely different than the measured particle signal, ensure that the ladder range used for generating the best-fit curve are within one-to-two dilution steps away from the measured particle signal to increase accuracy.
47. Calculate the fluorophore concentration of each well using the best-fit curve above. Correct for sample dilution by dividing each well fluorescence concentration by 1/10 of the OD550 value determined in Step 42 to determine stock nM/OD550.
Critical Step: Particles were diluted 10-fold in Step 44. This factor needs to be corrected for the OD550 in the plate.
48. Average the nM/OD550 values from each well and report as the mean ± s.e.m. Calculate the surface-ratio between R:G signals using the equation below. The ratio of compDNAs is reflective of the ratio of the scaffold DNAs:
a. Ratio of R-nM/OD550 (R) to G-nM/OD550 (G)
1. If R > G then the ratio of R to G is (R / G):1
2. If R < G, then the ratio of R to G is 1:(G / R)
Microparticle size quantification
Critical: Particle size distributions are assessed using microscopy imaging. We propose two methods: brightfield microscopy (Option A) and confocal microscopy (Option B). While brightfield requires less material preparation, confocal imaging of fluorescent particles produces defined silhouettes and reduces off-target quantification of debris.
Option A (Brightfield):
49. Pipette 10 µL of diluted particle solution from Step 31 (targeting ~5-10 OD550) onto a clear microscope slide and overlay a coverslip. Particles should be monodispersed to enable size quantification. If particles are too dense during imaging steps, dilute appropriately.
50. Visualize the microparticles under brightfield microscopy. Illumination and exposure settings should be optimized such that particle core/edges appear dark relative to the background. Acquire at least five representative images.
Option B (Confocal):
51. Particles must first be hybridized using saturating levels of fluorescent compDNA as described in Steps 40-42 and 44. A small amount of particles are needed for imaging (~5-10 OD550 in 30 µL) so adjust starting particle amount to minimize particle waste.
52. Pipette 10 µL of diluted, fluorescent particle (target ~5-10 OD550) onto a clear microscope slide and overlay a coverslip. Seal the coverslip corners with clear nail-polish. After the corners have partially dried and flattened, seal the sides of the slips by connecting each corner with nail-polish. This will prevent sample drying and allow for slide-inversion on the microscope if needed.
53. Visualize particles under confocal microscopy. Adjust laser power and exposure settings for the relevant laser-line, careful to avoid photobleaching. Height-focus should be set using the fluorescence channel. Acquire at least five representative images.
54. Analyze images using ImageJ to determine particle diameters. Size distribution curves can be generated in software such as Graphpad.
Antibody conjugation with complementary DNA
Critical: This procedure describes the conjugation of antibodies with amine-labeled compDNA using an NHS-PEG-Mal linker at a 2 mg antibody scale. This protocol does not change whether the DNA is labeled, but for most applications we recommend a dyeless-DNA. If a dye-labeled DNA is used, special attention should be placed to the charge of the dye; we have found that positively charged dyes may have increased association with the antibody and thus leads to purification difficulties. Ab-DNA can be labeled for quantification purposes after purification if required (see “Preparation of antibodies for surface loading quantification”).
55. Calculate the volume needed for 2 mg of antibody and prepare a Glen size-exclusion column that is appropriately sized for the antibody volume per the manufacturer’s instructions. Buffer exchange washes should be 10 mM EDTA in 1x PBS, Ca2+/Mg2+ free (PBS-EDTA).
Critical Step: Ensure that the buffer does not contain any amine-groups (e.g., Tris) as this will compete to react with NHS reagent used later.
56. Buffer exchange the antibody into PBS-EDTA per Glen column manufacturer’s instructions; collect into a new tube (reaction tube).
57. Measure the antibody A280 using Nanodrop with an appropriate dilution. Place the antibody at 4°C. Protein concentration can be calculated using the following equations:
b. (A280 * Dilution) / 1.33 = (mg/mL antibody)
c. ((mg/mL antibody) * 1000) / 155 = nmol antibody, where 155 is the antibody molecular weight (kDa).
58. Calculate volume of amine-compDNA needed for 4x molar excess relative to antibody in Step 57. Move this volume into a new tube (DNA reaction tube). The following equation can be used:
d. nmol DNA needed = 4 * (nmol antibody)
e. mL of DNA needed = (nmol DNA needed) / (µM DNA stock)
Critical Step: Ab-DNA conjugate yield can be improved by increasing the amount of DNA used, although this may require additional purification steps. The ratio of DNA-to-protein will need to be optimized for other biomolecules and linkers.
59. Calculate the mg of NHS-PEG-Mal for 20x molar excess relative to DNA from Step 58. Dissolve linker in a small volume of DMSO, with at least 30 µL per 0.8 mg of linker.
60. Add 20x molar-excess dissolved-linker to the DNA tube and incubate for 1 hour at 37°C. If the reaction DMSO volume exceeds 5% (v/v), add HEPES (100mM, pH 7.2) until 5% DMSO is reached.
61. When the DNA-PEG-Mal reaction from the previous step is nearly complete, dilute TCEP to 5 mM in PBS-EDTA. Calculate the volume of 5 mM TCEP needed for 4.5x molar excess relative to antibody amount determined in Step 57. Add this TCEP volume into the antibody tube and incubate for 1 hour at 37°C. Afterwards place the antibody at 4°C.
Critical Step: A lower molar excess can be used but may require longer incubation; longer timing or increased molar excess can result in different reduction cleavage products.
62. Precipitate the DNA as described in Step 3. During the precipitation, a second Glen column should be equilibrated to PBS-EDTA. The final volume after DNA precipitation will be 200 µL, so prepare an appropriately sized Glen column.
63. After 30 minutes at -20°C, centrifuge the DNA reaction tube at 18000 g for 10 minutes at 4°C. Remove supernatant and resuspend in 200 µL PBS-EDTA. Use the Glen column to buffer exchange to PBS-EDTA to remove any excess unreacted linker from the DNA-PEG-Mal.
64. Determine the DNA-PEG-Mal concentration from the Nanodrop A260 and Beer’s Law. Reference Table 1 for the relevant extinction coefficients.
65. Add 4x molar excess of DNA-PEG-Mal into the antibody tube and incubate for 1 hour at 37°C. Afterwards, place the antibody reaction tube at 4°C overnight.
Critical: The following steps are required for removal of free, unreacted DNA-PEG-Mal from the Ab-DNA conjugate which can compete for surface hybridization.
66. Use a ring-stand clamp to suspend a resin gravity column over a liquid waste container Assemble the column by placing the column filter at the bottom end nearest the exit port and capping the bottom. Vortex a bottle of Protein G resin beads and add 1.5 mL of the bead suspension followed by a sufficient volume of 1x Protein G binding buffer (binding buffer, diluted in water) to fill the column.
Critical Step: 1.5 mL of suspension results in ~0.75 mL column volume (CV) of resin after liquid drainage. Varied resin amounts can be used depending on the amount of protein being purified.
67. Remove the column cap and allow for the buffer to drain. When ~3/4 of the column height remains, cap the bottom, and wait 20 minutes for the resin to settle. Place a second column filter into the column and push until above the binding resin. Do not trap bubbles beneath the filters as this can slow the elution of the column.
68. Add 5 CVs (~3.75 mL) of binding buffer and allow to drain. Remove the waste container under the column and replace with a 15 mL capture conical tube.
69. Remove the capture tube and replace with another 15 mL conical tube. Add the Ab-DNA from the first capture tube. Repeat twice more by loading the flow-through to ensure maximum column binding. Discard the last flow-through.
70. Place a waste container underneath the filter column and wash with 10 CVs of binding buffer. While the column is washing, label ~thirteen 1.5 mL tubes—five for the acidic elutions, three for the neutral, and five for the basic. Add 55 µL of 10x acidic elution neutralization buffer into each acidic elution tube and 55 µL 10x of basic elution neutralization buffer into each basic elution tube.
Critical Step: If different elution volumes are captured per tube, the volume of neutralization buffer should be adjusted to achieve a final 1x concentration.
71. Add 3 CV (2.5 mL) of acidic elution buffer into the column and begin capturing 500 µL of flow-through into each acidic capture tube. Mix each tube afterwards to ensure the neutralization buffer has mixed into the flow-through.
72. After all acidic buffer has passed, add 3 CV of binding buffer and capture a third of the volume into each of the neutral tubes.
73. After all binding buffer has eluted, add 3 CV of basic elution buffer and capture 500 µL of flow-through into each basic capture tube. Mix each tube afterwards to ensure the neutralization buffer has mixed into the flow-through.
74. Place a waste container underneath the column and add 5-10 CV of binding buffer. After draining, cap the bottom and add binding buffer to cover just above the top resin. Label and store at 4°C if subsequent purifications are needed.
75. Quantify the A260 and A280 of each elution tube using Nanodrop. Dispose all tubes where the A280 indicates minimal protein recovery (<5-10% of the original theoretical protein amount) and also dispose when the A280 / A260 ratio is less than 0.9.
Critical Step: This step is the most critical for improving the purity of the final Ab-DNA. The A280 / A260 ratio can slightly vary although the tubes which primarily contain the unbound DNAs should have a ratio much less than 1.0.
76. Dialyze the Ab-DNA with 1x PBS using a 50 mL dialysis column (10K MWCO) and place onto an orbital shaker at 4°C as per the manufacturer’s instructions. Swap the 1x PBS after 2 hours and 4 hours cumulative time. After the final swap, dialyze overnight. The next day, collect the Ab-DNA from the dialysis column and store at 4°C.
Critical Step: This step removes the glycine and other buffer components which may inhibit downstream quantifications and purifications. The glycine must be removed if additional Fc-affinity column purifications are needed, otherwise the antibody cannot bind to the resin.
77. Use a microBCA kit to determine the protein concentration (in mg/mL) within the Ab-DNA conjugate according to the manufacturer’s recommendations.
78. The DNA concentration within the Ab-DNA is required for hybridization calculations, but this requires additional steps to calculate since both the antibody and the DNA independently contribute to both A260 and A280. Refer to Box 2 to solve for the DNA concentration within the Ab-DNA.
Critical Step: If a dye-labeled Ab-DNA was used, then the A260 (DNA component of Ab-DNA) can be estimated on a plate spectrophotometer using a standard fluorescent curve of known compDNA-dye concentrations and comparing the fluorescence of a known dilution of Ab-DNA.
79. Use Urea-PAGE to confirm that free DNA has been removed from the Ab-DNA conjugate. Prepare dilutions of Ab-DNA and pure-DNA and run the gel according to Steps 13-18.
Critical Step: If a dye-less DNA was used for conjugation, Urea-PAGE must be performed to later stain the DNA with Sybr Gold which is not compatible with SDS-PAGE gels. If a dye-labeled DNA was used, then SDS-PAGE gel is recommended as the antibody bands are more clearly defined.
80. Calculate the Ab-DNA purity with the equation below. If the sample is not pure (e.g., purity <0.95), then the purification Steps 72-82 must be repeated before proceeding:
I. Ab-DNA Purity = (Intensity of Ab-DNA band) / ((Intensity of Ab-DNA band) + (Intensity of DNA band))
81. Prepare a sufficiently sized Glen column using 0.1 M triethylammonium acetate buffer (TEAA, pH 7.0) as the exchange buffer. Buffer exchange the Ab-DNA into the TEAA and aliquot into separate tubes for lyophilization. Label the estimated protein and DNA amount in each tube to calculate the new concentrations when later resuspending.
Critical Step: Lyophilization is critical for Ab-DNA long term storage and to concentrate to facilitate particle hybridization. Previously, spin-concentrator columns were used but this resulted in significant protein loss onto the concentrator membrane. This was more apparent when using DNAs labeled with charged fluorescent dyes.
Critical Step: TEAA is a volatile buffer and thus does not leave salts after lyophilization, which could damage the proteins at high concentrations. Trehalose can be added as a cryoprotectant.
82. Freeze the Ab-DNA using liquid nitrogen as described in Step 33 and lyophilize overnight. The next day, resuspend an Ab-DNA aliquot in 0.1 µm-filtered PBS so that the concentration of the antibody is between 3 to 6 uM, using the concentrations determined in Steps 80 and 81 to determine the new concentrations after resuspension. Store remaining aliquots at -20°C.
Critical Step: Higher resuspension concentrations (>1 mg/mL) are important for the stability of proteins and to have more reasonable volumes to work with during particle hybridization.
Pause Point: Lyophilized proteins are stable at -20°C for over 2 years. Antibody can remain stable at 4°C for over a year. The shelf-life of other proteins should be assessed and monitored.
83. (Optional): Sodium azide (0.05%) can be added to a desired concentration to limit microbial growth once resuspended and stored at 4°C.
Preparation of antibodies for surface loading quantification
Critical: Flow cytometry can be immediately used to verify the ratio between protein species on particle surfaces if the conjugated compDNA was labeled with a fluorescent dye. If unlabeled compDNA was used for conjugation, NHS-dye labeling of the antibody is first required. Below we describe the labeling of αCD28-compR. The procedure is identical for labeling αCD3-compG but with a different fluorophore. To reduce non-specific interactions between the Ab-DNA and the particle, we recommend using negatively charged dyes for Ab-DNA labeling.
84. For later quantification, record the A260 and A280 of the Ab-DNA. Calculate the ratios R1 = A280 / (µM antibody) and R2 = A260 / (µM DNA), where the respective antibody and DNA concentrations are known from Step 82 after resuspension from lyophilized stock.
85. Resuspend NHS-Alexafluor-488 (AF488) to 2 mM in DMSO. Aliquot ~50 ug (0.32 nmol) of purified αCD28-compR (~100 µL at 0.5 mg/mL). Add 8x molar excess of 2 mM AF488 into the antibody aliquot and react for 1 hour at 37°C. Record the final reaction volume.
Critical Step: Do not exceed 5% DMSO (v/v) to reduce protein denaturing. To prevent this, either make a more concentrated stock of NHS-dye or dilute with HEPES (100mM, pH 7.2).
86. Prepare an appropriately sized Zeba spin desalting column according to the manufacturer’s instructions, replacing the buffer with 1x TE. Load the αCD28-compR-AF488 onto the column and spin as recommended.
87. Measure the A260, A280, and A488 to solve for the antibody, DNA, and AF488 concentrations using the below equations:
i. µM antibody = A280 / R1, where R1 is from Step 84. Similarly, µM DNA = A260 / R2, where R2 is from Step 84.
ii. AF488 µM = A488 / (Extinction coefficient AF488).
iii. Number of AF488-dye per antibody = (µM AF488) / (µM antibody)
Critical Step: Depending fluorophore intensity and dilution, the absorbance may be greater than 1.0. If so, redo this step using a higher dilution. The number of AF488-dyes per antibody should be above 1.0 and can be used to indicate successful conjugation.
Particle surface loading of antibody
Critical: The following describes the loading and quantification of αCD28-compR-AF488 and αCD3-compG-AF647 DNA-conjugates onto microparticles presenting a 1:1 R:G DNA scaffold. These steps assume NHS-dye labeled antibodies were prepared previously, although the procedure is identical for any fluorescently tagged antibody (e.g., using dye-labeled DNA during Ab-DNA synthesis)
88. Assuming particles are lyophilized in -20°C storage, prepare a small quantity of particles to allow for 1 OD550 in a final volume of 100 µL TE-Tween by following Steps 37-39 (adjusting for the desired OD550).
89. Assuming that antibodies have been prepared and labeled using NHS-dye, calculate the necessary volume of αCD28-compR-AF488 and αCD3-compG-AF647 to reach a final concentration of 30 nM each in 100 µL (total hybridization volume).
Critical Step: The antibody loading capacity on 1.5 µm particles is approximately 20 nM/OD550. Given that the scaffold ratio is 1:1, each antibody will maximally load ~10 nM/OD550. Since loading is at 3x excess of the theoretical limit, each antibody is hybridized at 30 nM/OD550 for a combined antibody concentration of 60 nM/OD550
Critical Step: Particle loading should occur between 1-10 OD550. The total reaction volume is flexible, although the total reagent use should be considered. If done under sterile conditions, particles can be used for biological applications using a small sample for flow analysis.
90. Refer to Table 4 for calculating the hybridization volumes, adjusting for the target OD550, antibody loading capacities, and antibody concentrations.
91. Spin the 100 µL of 1 OD550 particles for 5 minutes at 6000 g and remove supernatant until the calculated particle volume needed from Step 90 is reached.
92. To a 1.5 mL tube, add 50 µL of 2x hybridization buffer, the Ab-DNAs, and extra TE-Tween using the values in Step 90. Resuspend the particles and add into this reaction tube. Use the micropipette to mix and sonicate briefly (~5-10 seconds). Incubate for 30 minutes at 37°C.
93. Wash particles twice according to Step 41 at 4°C. After the last wash, resuspend in 500 µL TE-Tween.
Particle antibody loading quantification using flow cytometry
Critical: This procedure quantifies the microparticle surface loading of αCD28-compR-AF488 and αCD3-compG-AF647 using flow cytometry. Blank and single-antibody loaded particles are made using the method described in Steps 93-96 and are used for compensation controls and downstream calculations. Use single DNA-sequence scaffolded particles (R or G only) for single-color controls to saturate the surface with their respective antibody species. The plate spectrophotometer used in the previous section “Particle surface DNA loading analysis” can be used as an alternative quantification tool, although this uses prohibitively more material due compared to flow cytometry.
94. Perform flow cytometric analysis on particles from Step 93. Reference Box 3 for performing surface-loading analysis using software such as FlowJo
T cell enrichment from leukapheresis products
Critical: This procedure describes the isolation of either CD4+ or CD8+ T cells from leukapheresis blood product using commercial negative selection beads.
95. In a sterilized BSC, isolate CD4+ or CD8+ T cells from leukapheresis blood using the EasySep Enrichment Kit per the manufacturer’s instructions. Wash steps should be performed using sterile-filtered PBS-FBS wash buffer (see “Reagent Setup”). When required, cells should be centrifuged at 300 g for 5 minutes at 4°C.
96. After cells have been enriched, spin down the cells at 300 g for 5 minutes at 4°C. Calculate the volume to resuspend cells between 10-50 x 10^6 cells/mL. Remove the supernatant and resuspend in sterile freezing medium to the desired concentration. Aliquot 1 mL of cells into each liquid nitrogen compatible freezing vial and place into a polyethylene CoolCell. Immediately transfer the CoolCell into the -80°C freezer overnight. Transfer freezing vials to liquid nitrogen storage the following day.
Pause point: T cells can be stored in liquid nitrogen for over a year and thawed when needed.
T cell expansion using ICEp
Critical: This procedure describes CD4+ T cell culturing using ICEps which is identical for CD8+ T cells. Cells will be expanded in a 96-well (flat-bottom) culture plate throughout, although they can be transferred to larger well-plate volumes as long as the appropriate cell concentrations are maintained. ICEps should be prepared with αCD3 and αCD28 one day before T cell activation as described in the previous section, “Particle surface loading of antibody”. The quantity of particles required should be determined before T cell activation to reduce material waste. Complete T cell media (media) should contain 100 U/mL hIL2.
97. Centrifuge αCD3 and αCD28 loaded ICEps at 6000 g for 5 minutes at 4°C. In a BSC, carefully remove supernatant and resuspend to 1 OD550 (~20E6 particles/mL) in media. OD550 can be measured to verify desired particle concentration.
Critical Step: We will seed 25,000 T cells per 96-well, so 1.25 µL of particles (at 1 OD550) will eventually be added to each well for 1x particle to cell excess. Additional particle amounts can be added, although the total well volume should stay consistent between conditions.
98. Warm media in a 37°C water bath. Aliquot 9 mL of warmed media into a 15 mL tube. Remove a CD4+ enriched T cell vial from liquid nitrogen storage and thaw in the water bath. Just prior to fully thawing, move the vial into the BSC. Gently pipette to resuspend the cell pellet and transfer the volume into the 9 mL of warmed media to dilute the DMSO. Spin cells at 300 g for 5minutes at 4°C.
99. Remove the supernatant and resuspend cells in 10 mL of media and count the cells. Depending on the number of T cell conditions, dilute an appropriate volume of cells in media to ~0.278E6 cells/mL. After mixing, pipette 90 µL of cells per well in a 96-well plate.
100. Thoroughly mix the ICEps without generating bubbles and add an appropriate volume to each well (1.25 µL of 1 OD550 for 1x particle-to-cell). Occasionally resuspend stock ICEps to prevent particle settling. Add additional media for a total well volume of ~100 µL. The cells are now approximately at 0.25E6 cells/mL.
101. Using a multichannel pipette, gently mix all wells to thoroughly distribute ICEps and cells. Transfer the seeded culture-plate into a sterile incubator set to 37°C and 5% CO2.
102. After 24 hours (Day 1) visualize the plate under a bright-field microscope to observe cell clustering and look for any signs of contamination.
103. After 48 hours (Day 2), double the well volume using prewarmed media (~100 µL) by dispensing around the well perimeter, attempting not to disturb the cell clusters. Cells are typically not ready to be split at this day due to a freezing-related growth delay.
104. On day 4, resuspend the T cell wells and take a small sample for counting. Calculate the volume containing 25000 cells and reseed this volume into an unused well. Add media for a total well volume of 100 µL. Track the cell expansion fold between well-splitting and repeat every two days until growth slows or a pre-determined end-point has been reached.
Critical Step: At this concentration, a two-day splitting procedure lets cells expand upwards of 10-15 fold without filling the entire well. Other plating conditions requires different schedules.
105. At the experiment endpoint, stain, and fix cells for flow cytometry analysis.