I. Conjugate isotopes to BSA
Steps 1 to 11 (MaxPar Polymer Tube) and 12 to 23 (BSA-biotin Tube) are performed in parallel. This protocol is adjusted from Han et. al. (2018).
MaxPar Polymer Tube
1. Use one MaxPar tube per 200 μg of BSA-biotin. Spin down.
2. For each conjugation, add 95 μL buffer to each MaxPar tube and mix by pipetting.
3. Add 5 μL of each isotope solution to each MaxPar tube. Vortex briefly.
4. Incubate for 60 min at room temperature (RT). Vortex briefly every 20 minutes.
5. For each 3 kDa filtration unit add 400 μL of L buffer and label the tube.
6. Centrifuge filter units at 12,000 x g for 8 minutes and discard the flow-through.
7. After incubation with the MaxPar polymer, add 200 μL of L buffer to each MaxPar tube and transfer the 300 μL to the 3 kDa filter unit.
8. Centrifuge at 12,000 x g for 25 minutes each MaxPar-containing filter units and discard the flow-through.
9. To each MaxPar-containing filter unit add 400 μL of C buffer.
10. Centrifuge at 12,000 x g for 25 minutes the MaxPar-containing filter units and discard the flow-through.
11. Add 100 μL of C buffer to resuspend the metal-loaded polymer.
BSA-biotin Tube
12. Add 400 μL of R buffer to a 30 kDa filtration unit.
13. Centrifuge at 12,000 x g for 8 minutes the and discard the flow-through.
14. After 35 minutes passed from the incubation of step #4, pipet 800 μg of BSA-biotin into the 30 kDa filtration unit (this is enough material for 4 conjugations of 200 μg each). Centrifuge at 12,000 x g for 8 minutes the BSA-biotin filter units and discard the flow-through.
15. Add R buffer to a total volume to 400 μL.
16. Centrifuge at 12,000 x g for 8 minutes the BSA-biotin filter units and discard the flow-through.
17. Add 400 μL of R buffer.
18. Centrifuge at 12,000 x g for 8 minutes the BSA-biotin filter units and discard the flow-through.
19. Dilute the TCEP with 596 μL of R buffer to 4 μL of 0.5M TCEP (use always a new tube).
20. After the MaxPar-containing filter units start spinning in step #8, add 400 μL of diluted TCEP to each BSA-biotin filter unit. Vortex briefly, and incubate at 37 degrees C for exactly 30 minutes.
21. Centrifuge at 12,000 x g for 8 minutes and discard the flow-through.
22. Add 400 μL of C buffer to the BSA-biotin filter unit and centrifuge at 12,000 x g for 8 minutes.
23. Add 400 μL of C buffer to the BSA-biotin filter
Mix metal-loaded polymer with BSA-biotin tub
24. Mix the 100 μL of resuspended metal-loaded polymer (Step #11) to 100 μL corresponding BSA-biotin tube (Step #23) and transfer the mixture to a 50 KDa filtration unit.
25. Incubate at 37 degrees C for approximately 1.5 hours.
26. Add with 200 μL of PBS, centrifuge at 12,000 x g for 8 minutes and discard flow-through.
27. Wash with 400 μL of PBS, centrifuge at 12,000 x g for 8 minutes and discard flow-through. Repeat four times.
28. Then add another 100 μL of PBS, mix by pipetting and rinse the walls of the filter. Invert the column into a new collection tube.
29. Centrifuge at 1000 x g for 2 minutes.
30. Using Nanodrop, measure the concentration of BSA-biotin and dilute it to 0.2 mg/ml in PBS containing sodium azide or Candor Antibody Stabilization Solution.
31. Store at 4 oC.
II. Load 3 μm beads with proteins (Avi-tagged proteins)
1. Aliquot 0.2 mL of 3 μm beads and add 10 μL of CSM-T. Mix by pipetting. Centrifuge for 1 min 21,000 RCF at RT, rotate tube(s) 180º and centrifuge again, 2 min 21,000 RCF at RT. Remove supernatant.
2. Resuspend in 200 μL CSM-T. Centrifuge for 1 min 21,000 RCF at RT, rotate tube(s) 180º and centrifuge again, 2 min 21,000 RCF at RT. Remove supernatant. Repeat wash once more.
3. Prepare a 1:20 dilution of each Protein-Avi (Avi-tagged proteins; stock at 0.25 g/L) by adding water (quickly store protein stock at -20° C after usage).
4. Further dilute protein-Avi from step #3 to desired molarity. For example, dilute Spike S1 by taking 6 μL protein to 4 μLCSM-T to generate a volume of 10 μL.
5. Resuspend beads from step #2 in 165 μL CSM-T. Then split in twenty tubes (7.5 μL each) labeled as "1", "2", "...", and "20".
6. Add 7.5 μL of the protein-AVI mixture (step #4) to each tube of beads (step #5) and mix by pipetting. You will have twenty tubes with 15 μL each.
7. Incubate 30 minutes at RT. Centrifuge for 1 min 21,000 RCF at RT, turn tube(s) 180º and centrifuge again, 2 min 21,000 RCF at RT.
8. Wash each tube with 100 μL CSM-T. Centrifuge for 1 min 21,000 RCF at RT, turn tube(s) 180º and centrifuge again, 2 min 21,000 RCF at RT. Repeat wash once.
9. Add 165 μL CSM-T to each of the 20 tubes. Store at 4 °C until use.
Prepare 20-barcodes plate (isotopes 159 to 164)
10. Prepare 20-barcodes plate by following the order described below:
a. In a 96-well V-bottom plate, prepare a group of 5 x 4 wells (row x column) with 94 μL of CSM-T. Label 1 to 20 from left to right and top to bottom.
b. For BSA-biotin-159 add 2 μL of a 1:10 dilution (prepare 3 μL BSA-biotin in 27 μL CSM-T) to the plate prepared in step #10a, add to wells: 1-10.
c. For BSA-biotin-160 add 2 μL of a 1:10 dilution (prepare 3 μL BSA-biotin in 27 μL CSM-T) to the plate prepared in step #10a, add to wells: 1-4, 11-16.
d. For BSA-biotin-161 add 2 μL of a 1:10 dilution (prepare 3 μL BSA-biotin in 27 μL CSM-T) to the plate prepared in step #10a, add to wells: 1, 5-7, 11-13, 17-19.
e. For BSA-biotin-162 add 2 μL of a 1:10 dilution (prepare 3 μL BSA-biotin in 27 μL CSM-T) to the plate prepared in step #10a, add to wells: 2, 5, 8, 9, 11, 14, 15, 17, 18, 20.
f. For BSA-biotin-163 add 2 μL of a 1:10 dilution (prepare 3 μL BSA-biotin in 27 μL CSM-T) to the plate prepared in step #10a, add to wells: 3, 6, 8, 10, 12, 14, 16, 17, 19, 20.
g. For BSA-biotin-164 add 2 μL of a 1:10 dilution (prepare 3 μL BSA-biotin in 27 μL CSM-T) to the plate prepared in step #10a, add to wells: 4, 7, 9, 10, 13, 15, 16, 18-20.
h. When the 6 BSA-biotin-metal are dispensed, mix each well by pipetting gentle up and down (carefully to not make bubbles). Store plates at 4 °C until use.
Prepare beads with 20-barcodes
11. In a 96-well V-bottom plate prepare a group of 4 x 5 wells (row x column) with 160 μL of beads from step #10. Label 1 to 20 from left to right and top to bottom.
12. Pipette 4 μL from each well from step #10 into each corresponding well in step #11 and incubate for 30 min at RT. Centrifuge for 5 min 2800 RCF at RT in a plate centrifuge. Remove supernatant.
13. Wash each well in 160 μL CSM-T. Centrifuge for 5 min 2800 RCF at RT in a plate centrifuge. Remove supernatant. Repeat wash once.
14. Resuspend each well in 50 μL CSM-T. Store plate at 4 °C until use.
Prepare 924-barcodes plate
15. For each of the 12 BSA-biotins (isotopes 165 to 176) do a 1:10 dilution by preparing one Eppendorf with 70 μLBSA-biotin stock and 630 μLCSM-T. On each of the wells, we dispense 1 μL of the 1:10 dilution.
16. Using TEMPEST, dispense 44 μLCSM-T to each well of the 10 96-well plates used for the 924 barcoding.
17. Run a TEMPEST generation dispensing 6 out of 12 BSA-biotins (isotopes 165 to 176) to each well to generate the 924 barcodes. Store plates at 4 °C until use.
Mix spike-loaded beads with 924-barcodes
18. Pool all wells of the 20-barcode beads (step #14) in a single tube (about 1 mL final volume).
19. Wash in 1 mL CSM-T. Centrifuge for 1 min 21,000 RCF at RT, rotate tube(s) 180º and centrifuge again, 2 min 21,000 RCF at RT. Carefully remove supernatant.
20. Resuspend beads from step #19 in 80 mL CSM-T. Place 20 mL to each channel of a 4-well reservoir.
21. Using a 96-well pipettor (or liquid handler) dispense 50 μL of beads from step #20 (barcoded beads with protein) to each well of the 10 v-bottom plates from the 924-barcoding (step #17). Incubate 2 hours at RT. Store at 4 °C and use next day.
Wash plates
22. Add 150 μL CSM-T to each well in all plates. Centrifuge for 1 min 21,000 RCF at RT, rotate tube(s) 180º and centrifuge again, 2 min 21,000 RCF at RT. Remove supernatant. Repeat wash once more.
23. Add 30 μL of CSM-T to each well.
Heat inactivate samples and dilute sample
24. Heat inactivate samples in a water bath at 55° C for 1 hour. Perform this step only once.
25. Distribute the samples to the plates. A maximum of 924 samples can be tested.
26. Dilute samples 1:10 by taking 10 μL of sample and resuspending in 90 μL of CSM-T.
A) Two-Bead Assay
27. Add in each well 10 μL of diluted sample from step #26 to each well of the 10 plates from step #23.
28. Mix with samples by pipetting using a 96-well liquid hander and incubate 30 min at RT (no shaking).
29. Add 100 μL CSM-T to each well in all plates. Centrifuge for 3 min 2800 RCF at RT in a plate centrifuge. Remove supernatant. Repeat wash once more.
30. Add 50 μL CSM-T to each well in all plates.
a. (Optional) Add 50 μL of diluted protein target to each well. Prepare 40 μL of the protein target (2 μg/μL) in 20 ml CSM-T. Incubate for 10 minutes.
b. Centrifuge for 5 min 2800 RCF at RT in a plate centrifuge. Remove supernatant. Add 50 μL CSM-T to each well in all plates.
31. Combine all samples into a 50 ml tube.
32. Centrifuge for 5 min 2800 RCF at RT. Discard supernatant.
33. Resuspend in 1 mL CSM-T. Keep ~10% as baseline (100 μL).
34. To the remaining sample (900 μL) add 450 μL of anti IgG MACS microbeads.
35. Incubate for 1 hour by mixing on a rotator.
36. Prepare two MACS LS columns on the magnet and prewash them with 1mL CSM-T.
37. Add 650 μLCSM-T then add 1 mL of beads to each MACS LS column attached on the magnet. Collect the flow-through into a 5 mL protein low-bind tube.
38. Add another 4 mL CSM-T and collect the flow-through into the same 5 mL protein low-bind tube.
39. Centrifuge for 5 min 2800 RCF at RT. Discard supernatant.
40. Resuspend pellet to 1 mL CSM-T combine to a 1.5 mL protein low-bind tube and centrifuge for 3 min 21,000 RCF at RT. Discard supernatant.
41. Resuspend pellet to 1 mL CSM-T and centrifuge for 3 min 21,000 RCF at RT. Discard supernatant.
42. The beads were washed three times with 0.5 mL H2O. Each time centrifuge for 3 min 21,000 RCF at RT and discard supernatant.
43. Resuspend the beads to 0.8 x 106 beads/mL H2O.
44. Run mass cytometer at 500 to 1000 events per second.
B) Wash free assay
27. Add in each well 10 μL of diluted sample from step #26 to each well of the 10 plates from step #23.
28. Mix with samples by pipetting using a 96-well liquid hander and incubate 30 min at RT (no shaking).
29. Add 40 μL of 3.2% PFA to 40 μL of sample to make final concentration of PFA 1.6% and incubate for 15 min.
30. Add 80 μL of 4% SDS to 80 μL of sample to make final volume 2% SDS and incubate 15 min.
31. Pool the samples in a reservoir and then transfer to a 50 mL tube.
32. Centrifuge at 2800 RCF for 5 min and discard supernatant.
33. Add 25 mL CSM-T, mix by inverting the tube and centrifuge at 2800 RCF for 5 min. Discard supernatant. Repeat once more.
34. Stain with 25 mL CSM-T containing 246 ng/L anti-IgG gold and 246 ng/L anti-IgM 115Ir antibodies and incubate for 30 min.
35. Centrifuge at 2800 RCF for 5 min and discard supernatant.
36. Add 200 μLCSM-T, mix by pipetting, transfer to a protein low-bind tube, and centrifuge at 21,000 RCF for 5 min. Discard supernatant. Repeat twice.
37. Wash beads three times with 0.5 mL H2O. Each time centrifuge for 3 min 21,000 RCF at RT and discard supernatant.
38. Resuspend the beads to 0.8 x 106 beads/mL H2O.
39. Run mass cytometer at 500 to 1000 events per second.