Microgel fabrication and tethering of SA-PD-L1

1.    Take out PEG-4MAL, Peg Biotin, and DTT from -20C refrigerator and place in desiccator container ~20 min prior to weighing.

2.    Make a 10 mM DPBS/HEPES solution (sol1).

3.    Make a 2% Span80/mineral oil solution (i.e. to 40 mL of mineral oil add 800 ul of span80). Make sure to use wide office pipette tips to pipette span80.

4.    Vortex 2% Span80/mineral oil for 1 min at high speed.

5.    Weigh out the appropriate amount of 20 kDa PEG-4Mal (15 mg), and Peg Biotin (2 mg), DTT (30 mg) into 1.5 mL Eppendorf tubes. Make sure to clean spatula with ethanol and wipe it completely, and spray gloves with ethanol to avoid static interactions while weighing.

6.    Resuspend peg Biotin in sol1 to make a 2 mM solution (i.e. for 2 mg add 1 ml of sol1), this makes sol2.

7.    Resuspend 20 kDa PEG-4Mal in sol2 to make a 5%w/v solution (i.e. 5 mg in 100 ul) this makes sol3. Solution needs to incubate for at least 15 min.

8.    Reconstitute DTT in sol1 to 30 mg/mL (i.e. for 30 mg add 1 mL of sol1).

9.    Aliquot 5 mL of 2% Span80/mineral oil in a 15 mL conical tube and add 395 ul of DTT solution to the tube.

10.                Vortex for 1 min at high speed.

11.                Place in sonicator for 15 min with no heat. In the meantime prepare work station with pumps and microfluidic device. Prime microfluidic device by flushing it with a full 1ml of 2% Span80/mineral oil.

12.                Set pumps to:

a.     Oil: 1 ul/min

b.     PEG: 5 ul/min

c.     DTT cross linker: 35 ul/min

13.                After 15 min load sol3 to peg line using 1 ml Hamilton syringe, use a withdrawal rate of 50 ul/min, make sure to switch back to inject after done.

14.                Load oil to oil line using 1 ml Hamilton syringe.

15.                After sonication load DTT solution to BD plastic 5 mL syringe.

16.                Run solution through microfluidic flow-focusing device and collect microgels in a 15 mL conical tube filled with 10 mL of cold dPBS.

17.                After running through all the PEG stop pumps, and place 15 mL conical in mini centrifuge in the new TC room.

18.                Centrifuge at 600g for 5 min.

19.                Remove fluid up to 2 ml line with a glass pipette, and add new fresh 12 mL of cold dPBS.

20.                Repeat steps 21-22 about five times to remove oil.

21.                Store microgels in 10 ml of DPBS 1x at 4C for up to a week.

22.                For attaching immunomodulatory ligands, first, coat a 15 mL tube with 30% FBS for 15 min.

23.                Rinse twice with 1x dPBS.

24.                Using a coated pipette tip remove the desired number of microgels to be conjugated from the original vial and place in a coated 15 mL tube.

25.                If volume >2 mL centrifuge as in step 21, and remove supernatant until you have 1.5-2 mL left in the tube.

26.                Add ligand at the desired concentration (typically 1000 microgels: 1ug protein)

27.                Incubate at RT for 2 hrs in a rotator.

28.                After two hours repeat step 21.

29.                Remove ~ 1 mL of supernatant and store at -20C. This will be used for western blot to assess capturing efficiency.

30.                Add 12 mL of dPBS and mix.

31.                  Centrifuge at 600g for 5 min.

32.                Repeat 33-34.

33.                Remove supernatant up to the microgel line or as much as possible without disturbing the pellet.

34.                Microgels are ready for transplant (within 1-5 hrs). Do not store, discard leftover microgels.

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