Mass cytometry staining for human bone marrow clinical samples

Summary This protocol details a staining technique optimized for immunophenotyping of human bone marrow immune populations using mass cytometry. The protocol accounts for the limitations of working with human bone marrow, such as reduced viability, low cell counts, and fragile cell pellets, to successfully acquire single viable cells ready for downstream analysis. This assay can be used to characterize the activation, exhaustion, and cytotoxicity of immune populations and ensure comprehensive immunophenotyping of human bone marrow clinical samples.

To isolate bone marrow mononuclear cells (BMMC), BMMCs are first enriched from aspirates by density gradient centrifugation using Ficoll-Paque within SepMateä PBMC Isolation Tubes. Any remaining erythrocytes are then briefly lysed using 13 RBC Lysis Buffer, and samples are washed with 13 PBS to remove residual lysis buffer and any cellular debris. 4. Pre-wet a 100 mm MACSâ SmartStrainer with 13 PBS within a 50 mL conical tube. Discard 13 PBS used to pre-wet MACSâ SmartStrainer from your 50 mL conical tube 5. Slowly filter bone marrow sample through the pre-wet MACSâ SmartStrainer into 50 mL conical tube, using the end of a syringe plunger to break apart any clots above the filter and to filter out any bone debris 6. Make note of the volume of bone marrow aspirate post-filtering, then dilute bone marrow sample with 13 PBS three times its volume (for example, dilute 10 mL of bone marrow with 30 mL PBS) 7. Mix the 13 PBS and bone marrow by gently inverting the tube 8-10 times 8. Add 15 mL of Ficoll-Paque to a 50 mL SepMateä tube by carefully pipetting it through the central hole of the plastic insert. The top of the density gradient medium should be just above the insert Note: Any large bubbles present within the density gradient medium should be removed by spinning at 300 g for 1 min prior to loading your sample.
9. Gently layer the diluted bone marrow onto the Ficoll-Paque by pipetting it down the side of the SepMateä tube. The bone marrow should overlay on top of the Ficoll-Paque.
Note: If the volume of diluted bone marrow is larger than 30 mL, prepare multiple SepMateä tubes and evenly distribute the sample CRITICAL: Do not let the Ficoll-Paque and bone marrow aspirate mix under the plastic insert within the SepMateä tube, as this will affect the yield, purity and 127I content of the isolated BMMCs.
10. Centrifuge the SepMateä tube(s) containing the diluted bone marrow aspirate and Ficoll-Paque at 1200 g for 20 min at room temperature, with the brake on 11. Carefully pour the top layer above the plastic insert from the SepMateä tube(s) (which contains enriched BMMCs) into a new 50 mL conical tube CRITICAL: Do not hold SepMateä tube in the inverted position for longer than 2 seconds, as this will cause separated RBCs to pass through the plastic insert into the new 50 mL conical tube.
12. Discard the SepMateä tube containing RBCs and granulocytes in biohazard waste 13. Centrifuge the new at 300 g for 8 min, with the brake on 14. Pour off the supernatant and resuspend the cell pellet in 20 mL of 13 RBC Lysis Buffer, and gently invert the tube 8-10 times 15. Immediately after centrifuge the 50 mL conical tube containing sample at 300 g for 8 min with the brake on 16. Pour off supernatant and resuspend the pellet in 40 mL PBS. Mix by gently inverting the tube 8-10 times 17. Centrifuge at 300 g for 8 min with the brake on 18. Pour off supernatant and resuspend pellet in 10 mL PBS 19. Count cells using 0.4% Trypan Blue (1:1 dilution, 10 mL sample in 10 mL Trypan Blue) with the Invitrogenä Countess II Automated Cell Counter (or an alternative cell counter) a. Remove 10 mL of resuspended sample and mix well with 10 mL 0.4% Trypan Blue b. 23. Mix by gently pipetting without introducing bubbles, and incubate at 4 C for 15 min 24. After 15 min, wash the BMMC and magnetic bead mixture by pipetting very gently with 2 mL autoMACS Running Buffer 25. Centrifuge samples at 300 g for 10 min, with the brake on 26. Aspirate the supernatant and resuspend the bead pellet in 500 mL autoMACS Running Buffer 27. Transfer the resuspended sample to a new 15 mL conical tube 28. Label 2 new additional 15 mL conical tubes as ''CD138+'' and ''CD138-'', followed by the sample ID 29. Place the 15 mL tube containing the re-suspended sample in the autoMACSâ Pro Separator tube rack in the designated sample slot, and place the empty pre-labeled 15 mL conical tubes in each of the appropriate locations for the positive and negative sample fractions to deposit into 30. Ensure there are adequate volumes of autoMACS Washing Buffer, autoMACS Running Buffer, 70% EtOH, and sufficient space in the waste in reagent reservoirs attached to the autoMACSâ Pro Separator 31. Select the protocol named ''Possel'' on the autoMACSâ Pro Separator to enable automated bead selection to begin 32. Once autoMACSâ Pro Separator is done with enriching CD138+ and CD138-cells fractions, centrifuge samples at 300 g for 8 min, with the brake on 33. Remove supernatant and resuspend each sample in 1 mL of PBS. 34. Count each cell fraction (CD138 + /CD138 -) using 0.4% Trypan Blue (1:1 dilution, 10 mL sample in 10 mL Trypan Blue) with the Invitrogenä Countess II Automated Cell Counter (or an alternative cell counter) a. Remove 10 mL of resuspended sample and mix well with 10 mL 0.4% Trypan Blue b. Load 10 mL of diluted of the Trypan Blue diluted sample to a Countess Cell Chamber 35. Dilute both CD138 + and CD138fractions to 10 mL total by adding 9 mL PBS to wash away remaining autoMACS running buffer 36. Centrifuge samples at 300 g for 8 min, with the brake on. 37. Carefully aspirate and discard supernatant.

MATERIALS AND EQUIPMENT
CRITICAL: Sodium Azide is carcinogenic and toxic if swallowed, inhaled, or if in contact with skin. Handle in a fume hood using personal protective equipment.
Note: The timing and reagent consumption in this protocol is relative to the handling of 20 samples.

STEP-BY-STEP METHOD DETAILS
Bone marrow thawing and cell counting

Timing: 1 h
To prepare the cryopreserved bone marrow samples for mass cytometry staining, the samples must first be thawed, counted, and viability assessed.
1. Before staining, fill the appropriate number of 15-mL conical tubes (one per sample) with 9 mL of RPMI +10% FBS +1% Antibiotic-Antimycotic (named Complete RPMI from now on) and place at 37 C 2. Prepare the surface antibody master mix in CyFACs (Table 1) 3. Thaw the samples by placing the frozen vial directly into the 37 C water bath until content is thawed, approximately 45 s, few small ice crystals might remain in the vial. 4. Transfer the totality of thawed cell suspension into the 15 mL conical containing 9 mL of 37 C complete RPMI 5. Count cells using AO/PI (1:1 dilution; 20 mL sample in 20 mL AO/PI) with the Cellometerâ Auto 2000 Cell Viability Counter a. Gently invert the conical containing the freshly thawed cells to ensure accurate cell counts b. Add 20 mL of the AO/PI-diluted sample to a Cellometerâ SD100 Cell Counting Chamber c. Cells should be >90% viable 6. Adjust concentration of cell suspension to 3 3 10 6 cells/sample in complete RPMI 7. Centrifuge at 400 g for 10 min a. While the sample is spinning down, prepare the 103Rh Viability Stain (1:500 dilution of 103Rh Cell-ID intercalator in 37 C warm complete RPMI) 8. After the sample is spun down, carefully aspirate and discard the supernatant 9. Resuspend the sample in 1 mL of 103Rh Viability Stain 10. Incubate for 15 min at room temperature 11. Once incubation is complete, fill conical up to 5 mL with complete RPMI and spin down (400 g, 10 min) 12. Carefully aspirate and discard supernatant without disturbing the cell pellet 13. Resuspend each cell pellet in 50 mL CyFACs and proceed to Fc-block and surface staining CRITICAL: If cell viability is below 90%, filter the samples using 70 mM cell pre-wetted strainers to obtain a single cell suspension.
Optional: The surface antibody master mix can be prepared during the viability stain incubation, but we recommend preparing the cocktail prior to the thawing of clinical samples to avoid any critical errors.

Fc-block and surface staining
Timing: 1 h This portion of the protocol stains the sample with pre-selected metal-tagged antibodies to be acquired and used in downstream analysis. Prior to antibody staining, the samples are Fc-blocked to prevent non-specific staining of antibodies.
14. Fc-block procedure: a. Add 5 mL undiluted Fc-block to each sample b. Incubate on ice for 10 min i. During the incubation, aliquot the prepared surface antibody cocktail and distribute 50 mL/ well according to 96 well plate map 15. Surface staining: a. Once the incubation is complete, distribute the totality of Fc-blocked samples into the appropriate wells (already containing the antibody cocktail) according to the plate map b. Incubate for 30 min at room temperature i. During incubation, prepare the eBioScience TM FoxP3 Fixation/Permeabilization Buffer as per manufacturer protocol (https://tinyurl.com/foxp3TF) (briefly, mix 1 part fix/perm concentrate (00-5123-43) + 3 parts fix/perm diluent (00-5223-56)) ii. During incubation , prepare the eBioScience TM Perm/Wash solution by diluting 2 mL of 103 Permeabilization Buffer stock into 18 mL of Ultra-Pure H 2 0 c. Pellet cells by plate centrifugation (400 g, 5 min) d. Carefully aspirate supernatant ( 100 mL), wash once with 200 mL CyFACs, spin plate down (400 g, 5 min) and proceed to intracellular staining Optional: The surface antibody master mix can be added individually to each sample well after the Fc-blocked cells have been plated, but this increases risk of technical error and experimental time.
Note: The FoxP3 Fixation/Permeabilization Buffer is prepared in excess and will be needed for the DNA stain at the end of the protocol.

Permeabilization and intracellular staining
Timing: 1.5 h This portion of the protocol stains the sample with pre-selected metal-tagged antibodies that target intracellular markers. Prior to intracellular staining, the samples are incubated with eBiosciences TM FoxP3 Fixation/Permeabilization Buffer to preserve cellular morphology while allowing the intracellular antibodies across the plasma membrane to stain intracellularly.  The final steps of the protocol stain the cell nuclei to allow for single-cell detection and discrimination by the Helios TM mass cytometer. After completing the DNA stain, the samples undergo a series of washes to prepare for acquisition.

EXPECTED OUTCOMES
At the end of this protocol, FCS files will be generated for every sample acquired on Helios TM and normalized using the FCS Processing tab of the Fluidigm CyTOF Software 7.0.8493. Prior to immunophenotyping, the FCS files must be cleaned as described in Figure 1 (Bagwell et al., 2020) (Thrash et al., 2020). Briefly, cell events are gated by event length versus time, beads (to remove EQ calibration beads), and Gaussian parameters (Residual, Width, Offset). Single cells are determined by a bi-axial plot of both DNA Intercalator channels. Specific to bone marrow, we identified live, CD45+ cells, and then confirmed the depletion of CD138 before continuing analysis. As noted in Figure 2, CD45 bone marrow populations are donor-dependent and can vary from a CD45 smear to distinct CD45 hi and CD45 dim populations. Further analysis focused on the CD45 hi lymphocyte population, excluding the non-viable and CD45 dim erythroid populations (Cloos et al., 2018).

LIMITATIONS
This protocol was established for staining extracellular proteins and secreted proteins, including cytokines, and other intracellular antigens. It is not applicable for staining of activation-induced phosphorylated antigens.

OPEN ACCESS
TROUBLESHOOTING Problem 1 Low cell recovery at the end of staining protocol (step 41)

Potential solution
Do not use microcentrifuge 1.5 mL or 2 mL tubes for this protocol, always use 96 well, V-bottom plate. Do not discard supernatant through plate flicking, but instead aspirate the volume to discard using a multichannel pipette.

Problem 2
High level of 127-iodine contamination in your samples (step 9 and data analysis)

Potential solution
The most popular source of 127-Iodine contamination is Ficoll -Paque, as it contains sodium diatrizoate. It is crucial to carefully remove the immune cells layer without touching the Ficoll -Paque layer or use tubes with a separation membrane. If contamination still occurs, it might be reduced by 3 supplementary washing steps after cell thawing.

Problem 3
Staining seems to ''fade'' or ''streak'' over time on certain markers (step 25 and data analysis)

Potential solution
Fixation using freshly prepared PFA is crucial. The fixative agent in the Fix/Perm buffers may degrade over time after repeated exposure to oxygen; freshly prepared PFA assures proper fixation and maintenance of staining signal. We suggest freshly fixing stained cells using 4% PFA. The concentration can be adjusted depending on specific needs of the assay, but not decreased more than 1.6% PFA.

Problem 4
Significantly more than 20 samples to stain and to acquire on mass cytometer (steps 1 and 42)

Potential solution
Up to 80 samples can be stained within the same plate, and the plate can be frozen after the 191-Ir DNA Intercalator incubation in freezing medium containing 90% of FBS and 10% of DMSO (Sumatoh et al., 2017). For long acquisitions, the best solution is to freeze all plates and book the mass cytometer for the number of consecutive days needed. To prepare samples for acquisition, thaw each plate the morning of acquisition, and continue with washes, treating the samples as if they were incubated overnight in a DNA-intercalator.
Problem 5 103Rh Viability Staining seems not to perform when analyzing data (step 9 and data analysis)

Potential solution
Be careful when modifying fixation and permeabilization buffers, as for example 103Rh viability staining is not compatible with methanol fixation. If other fix/perm reagents are being used, staining with Cell-IDä Cisplatin, Fluidigm #SKU 201064 might replace 103Rh Viability staining. Cisplatin binds covalently to protein and labeling remains strong through subsequent steps of the protocol.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Joanna Baginska, joanna_baginska@dfci.harvard.edu.