Murine brain tumor microenvironment immunophenotyping using mass cytometry

Summary Here, we present a mass cytometry protocol optimized to examine the phenotype of immune cells within the mouse glioma microenvironment, using a Sleeping Beauty transposon-mediated mouse glioma model. We describe antibody conjugation and titrations for analysis of immune cells. We then detail mouse brain tumor tissue collection and processing, staining, followed by data acquisition, analysis, and gating strategy. This protocol can be applied to any brain tumor-harboring mouse model. For complete details on the use and execution of this protocol, please refer to Alghamri et al. (2021).

3. Upon completion of the 30 min antibody incubation, purify the partially reduced antibody.
a. Add 300 mL of C-Buffer to the 50 kDa filter to wash. b. Centrifuge at 12,000 3 g at 25 C for 10 min in a microcentrifuge and discard the flowthrough. c. Add 400 mL of C-Buffer to the 50 kDa filter. d. Wait 15-20 min to allow antibody and polymer prep timing to align in an upcoming step. e. Centrifuge the reduced antibody at 12,000 3 g at 25 C for 10 min in a microcentrifuge. 4. Perform the polymer wash.
a. Add 200 mL of L-Buffer and the 100 mL polymer mixture to a 3 kDa filter. b. Centrifuge at 12,000 3 g at 25 C for 25 min in a microcentrifuge and discard the flowthrough. This table lists the metal conjugated antibodies used for the immunophenotyping in this protocol. The metals listed in the table are Neodymium (Nd), Samarium (Sm), Europium (Eu), Gadolinium (Gd), Dysprosium (Dy), Ytterbium (Yb), Erbium (Er), Praseodymium (Pr), Terbium (Tb), Holmium (Ho), Thulium (Tm), Lutetium (Lu), Bismuth (Bi), and Yttrium (Y). a Indicates the antibodies that are not pre-conjugated, so these must be conjugated in-house from purified antibodies.
c. Add 400 mL of C-Buffer and centrifuge at 12,000 3 g at 25 C for 30 min in a microcentrifuge.
Note: The C-Buffer is a part of the Maxparâ X8 Antibody Labeling Kits specified in the key resources table. This buffer is used in this protocol without any further modification.
Note: To save time, step 4 can be performed simultaneously with steps 2 and 3.
5. Conjugate the antibody. a. Retrieve the 3 kDa and the 50 kDa filters from the microcentrifuges and discard the flowthroughs. b. To reach a final volume of approximately 80 mL, add 60 mL C-Buffer to the residual volume in the 3 kDa filter and resuspend the polymer by rinsing the filter walls. c. Transfer the approximately 80 mL polymer solution from the 3 kDa filter to corresponding antibody solution in the 50 kDa filter.
Note: Accounting for the residual volume in the 50 kDa filter, the final volume should now be approximately 100 mL.
d. Rinse the walls of the 50 kDa filter and mix the solution gently by pipetting. e. Incubate at 37 C in a water bath for 90 min. 6. Wash conjugated antibody.
a. Add 200 mL of W-Buffer to the 100 mL conjugated antibody mixture within the 50 kDa filter and mix gently by pipetting.
Note: The W-Buffer is a part of the Maxparâ X8 Antibody Labeling Kits specified in the key resources table. This buffer is used in this protocol without any further modification.
b. Centrifuge at 12,000 3 g at 25 C for 10 min in a microcentrifuge and discard the flow-through. c. Repeat steps 6a and 6b three more times. 7. Determine yield.
a. Add 80 mL of W-Buffer to the 50 kDa filter to resuspend the conjugated antibody to a final volume of 100 mL. b. Rinse the walls of the filter and gently mix the W-Buffer and conjugated antibody by pipetting approximately five timesusing a 200 mL pipette tip. c. Using W-Buffer as the blank solution, quantify yield using the NanoDropä by measuring absorbance at 280 nm.
Note: In addition to the absorbance, the NanoDropä will return a concentration in ng/mL. Given that the total solution should be in 100 mL, calculate the total mass of conjugated antibody. Expected yield is about 60%, which can be calculated by dividing the mass of conjugated antibody by the 100 mg that were initially added in step 2c. 8. Store conjugated antibody in its final solution. a. Centrifuge 50 kDa filter at 12,000 3 g at 25 C for 10 min in a microcentrifuge. b. Add the calculated volume of Antibody Stabilizer PBS supplemented with 0.05% sodium azide.
Note: Use approximately 20 mL less than calculated to account for the residual volume.
c. Invert the 50 kDa filter into a new collection tube and centrifuge at 1,000 3 g at 25 C for 2 min in a microcentrifuge. d. Label, seal with parafilm, and store the tubes with conjugated antibodies at 4 C.

Titration of antibodies and assessing signal spillover
Timing: 2-3 days Optimal generation of an antibody panel for mass cytometry requires antibody titrations and checking for signal spillover. The data from the titration experiment is analyzed to identify the optimal concentration, at which, there is a good separation between antibody-labeled positive and negative populations. The spillover test is used to confirm that there is minimal overlap between antibody channels, as this can lead to inaccurate results and false conclusions. 9. Prepare the surface antibody tubes for the spillover test.
a. Label spillover tubes for the spillover test (''Panel A 1:50'', ''Panel B 1:50'', ''Panel C 1:50''), which correlate with the antibodies to be added to them (see Table 2). b. In the spillover tube labeled ''Panel A'', add 36 mL of Maxparâ Cell Staining Buffer and 1 mL of each antibody indicated in the column. c. In the spillover tube labeled ''Panel B'', add 39 mL of Maxparâ Cell Staining Buffer and 1 mL of each antibody indicated in the column except CD16/32 and CD206. d. In the spillover tube labeled ''Panel C'', add 40 mL of Maxparâ Cell Staining Buffer and 1 mL of each antibody indicated in the column. 10. Prepare the surface antibody tubes for the titration test.
a. Label titration tubes for serial dilutions (''Surface 1:50'', ''Surface 1:100'', ''Surface 1:200'', ''Surface 1:400'', ''Surface 1:800''). b. In the ''Surface 1:50'' tube, add 30 mL of Maxparâ Cell Staining Buffer and 2 mL of each antibody indicated in the column except the highlighted CD16/32 and CD206. Mix well. c. Add 50 mL of Maxparâ Cell Staining Buffer to the four other titration tubes. d. Perform a serial dilution by adding and mixing 50 mL of the 1:50 tube to the 1:100 tube; then transfer 50 mL from the 1:100 tube to the 1:200 tube, and so on until the 1:800 tube is left with a final volume of 100 mL of surface antibody mix (See Figure 1)  Note: The Panel A and Panel C tubes will be receiving a normal Fc Block solution (consisting of anti-CD16/32), so this antibody will be prepared in the same tube in step 13b. The Panel B tube will be receiving the lanthanide-labeled antibody targeting CD16/32 to examine this The table outlines the metal conjugated antibodies to be added to each tube during the test for assessing signal spillover. The CD16/32 and CD206 (MMR) antibodies are stained at times seperate from the rest of the antibodies. The CD16/32 antibody will be used during the Fc block staining step of the other two tubes. The CD206 antibody is used during the intracellular staining step.

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molecule on the cells, since this molecule is a target in the panel, so it will be prepared separately in step 13c.
b. In the ''Panel A&C Fc Block'' tube, add 2 mL purified anti-CD16/32 antibody (1 mg/mL) to 98 mL of Maxparâ Cell Staining Buffer to make the 1:50 solution at 0.02 mg/mL. c. In the ''Panel B Fc Block'', add 1 mL of the lanthanide conjugated anti-CD16/32 antibody (0.5 mg/mL) to 49 mL of Maxparâ Cell Staining Buffer to make the 1:50 solution at 0.01 mg/mL. 14. Prepare the Fc receptor blocking solutions for the titration tubes. 16. Prepare a cell suspension of naïve wild type mouse splenocytes in a concentration of 3 3 10 6 cells/mL of media. 17. Add 1 mL of splenocyte solution into 8 polypropylene tubes. Discard the rest. 18. Label these tubes as the spillover tubes (Panel A-C) and titration tubes (Titration 1:50-1:800). 19. Spin down the splenocytes at 300 3 g for 5 min at 4 C. 20. Resuspend the cells at 2 3 10 7 cells/mL of media. 21. Follow the ''cisplatin (cell viability) staining'' section protocol below. 22. Centrifuge the cells at 300 3 g for 5 min at 4 C and discard the supernatant. 23. Resuspend the pellets in 50 mL of the Fc receptor blocking solution which corresponds to each tube. 24. Incubate at 25 C for 10 min. 25. Wash the cells.
a. Add 2 mL of Maxparâ Cell Staining Buffer. b. Centrifuge at 300 3 g at 4 C for 5 min. c. Discard supernatant. 26. Gently pipette to resuspend the pellet in 50 mL of the surface antibody mix which corresponds to each tube (see Table 3). 30. Follow the ''fixing and intracellular antibody staining'' section protocol below using the intracellular antibody mix which corresponds for each tube (see Table 3).
Note: Since spillover tubes for Panel A and Panel C do not have the stains for intracellular antibodies, add Maxparâ Perm S Buffer with no antibodies in place of intracellular antibody stain.

Follow the ''fresh fix and DNA intercalator staining'' section protocol below.
Pause point: The cells can be left up to 2 days at 4 C until acquisition.
32. Follow the ''sample acquisition on CyTOFâ instrument'' protocol below. 33. Follow the ''normalization'' section protocols below. 34. Using Cytobank, as described in the ''expected outcomes'' section, gate for the cell populations using the sample antibodies for each concentration.  Table 2). There is no spillover if the cells stained with each antibody panel are only positive for the antibodies they were stained for. If samples are identified as positive for antibodies that were not added in that panel's antibody mix, there is most likely spillover. In this case, you would need to use a lower concentration of antibodies that are of the same element and adjacent atomic weight within that panel.
Note: For example, there would be spillover if a sample stained with ''Panel B'' antibody mix has cells that are positive for 143Nd, since the 143Nd-labeled antibody is not present in ''Panel B'' (see Table 2). Thus, you would need to use a lower concentration of the 142Ndlabeled and 144Nd-labeled antibodies in the main experiment to minimize their spillover into the 143Nd channel, since the 142Nd-labeled and 144Nd-labeled antibodies in ''Panel B'' are spilling over into the 143Nd channel.
36. Compare the ability of the antibodies to bind and identify the positive cell population using the different antibody dilutions (See Figure 2). The lowest antibody concentration that allows for clearly discerning the positive and negative populations of the antibody-targeted molecule should be selected and used for the main experiment.
CRITICAL: Give close attention to choosing the optimal dilution for each antibody. This determines the antibody dilutions to use for the remainder of the protocol.

Processing the tissues into the single cell suspension
Timing: 2.5 h (this time is for processing 6 mouse samples) This step details the process of generating a single cell suspension from the glioma-harboring GEMM and isolating the immune cells for staining.
37. Prepare materials. a. Per sample label: one 50 mL conical tube; two 15 mL conical tubes; one 70 mm cell strainer and one cryovial. b. Place the cell strainer into the 50 mL conical tube. c. Wet the cell strainer by adding 2 mL of media. d. Insert the conical tubes into an ice bucket. 38. Euthanize mouse and begin tissue collection.
a. Inside a fume hood, add 1 mL isoflurane to a cotton pad. b. Place the isoflurane-soaked cotton pad to the bottom of the Nalgeneä Polypropylene Desiccator chamber and cover that area with the desiccator plate. quickly cover the desiccator to prevent vaporized isoflurane from escaping the desiccator chamber. c. Place the mouse into the chamber and cover the chamber. d. Once mouse stops all movement, wait for 20 more seconds. e. Confirm the mouse is euthanized by toe pinch and perform decapitation.
Note: For isoflurane euthanasia, any chamber that allows for administration of highly concentrated, vaporized isoflurane without the mouse coming into direct contact with the liquid Figure 2. Example selection of optimal concentration of antibodies from titration data The titration step is used to determine the best concentration of antibody for use in the staining process for the main experiment. This CD11b, CD162, and CD4 titration data is an example of the gates generated from these antibodies at different concentrations. The blue arrow indicates it as the selected concentration for further use in identifying the positive cell populations. The red and blue boxes in the selected graphs identify the positive and negative cell populations, respectively. isoflurane can be used in place of the Nalgeneä Polypropylene Desiccator. Additionally, alternative forms of euthanasia, such as a cervical dislocation, may be used in place of isoflurane overdose as long as they do not elicit alterations in the cell characteristics.
39. Process the tumor to generate a single cell suspension.
a. Dissect the brain and place it onto a petri dish with enough media to cover the surface of the dish. b. Dissect the tumor mass under a stereo-microscope and place it on the 70 micron strainer.
Note: The GEMM tumors used in this protocol expressed GFP and were dissected based on fluorescence. Although not optimal, the dissection of tumors without a reporter can be conducted by location, appearance, and texture. Upon examining the area of the brain where the tumor cells were implanted, features such as changes in color and texture vary between healthy brain and tumors. These features can be used to dissect the murine brain tumors lacking reporters.
c. Dissociate the tumor by applying mechanical pressure with the end of a syringe plunger to push the tumor through the strainer. d. Rinse the strainer four times using 10 mL of media, making sure to keep the tube on ice. 40. Isolate the immune cells.
Note: Refer to ''materials and equipment'' section for instructions on how to prepare the SIP and media solutions.
b. Centrifuge the tumor cells at 300 3 g at 4 C for 5 min. c. Discard the supernatant and resuspend the pellet in 1 mL of media. d. Add the 1 mL cell suspension to 6 mL of media in a 15 mL conical tube labeled ''Tumor with Percoll''. e. Add 3 mL of the SIP solution and mix well. f. Slowly insert a 1 mL serological pipet filled with the 70% Percoll solution along the side of the tube until it reaches the bottom. g. Slowly layer 1 mL of 70% Percoll at the bottom of the tube.
CRITICAL: Exercise caution to very slowly add the 70% Percoll, pipetting too quickly will disturb the gradient and mix the layers.
h. Centrifuge the tube with the gradient at 800 3 g for 20 min at 25 C.
CRITICAL: This centrifugation step must be completed with the brakes off to prevent disrupting the gradient.
i. During centrifugation from the previous step, add 10 mL of media to a 15 mL conical tube labeled ''Tumor after Percoll''. j. Remove the tube from the centrifuge and use a transfer pipette to discard the top layers (containing debris, tumor cells, SIP-media solution) until it approaches the central whitecloudy immune cell layer located between the pink and clear layers (see Figure 3).
CRITICAL: Be careful not to disrupt the gradient when moving the tube.
k. Use another transfer pipette to remove the immune cell layer completely and resuspend it in the labeled ''Tumor after Percoll'' tube. l. Centrifuge the tube at 300 3 g for 5 min at 4 C. m. Discard the supernatant and resuspend in 1 mL of freezing media. n. Transfer to a labeled cryovial.

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o. Immediately place the cryovial in a -20 C freezer for one hour before transferring to a À80 C freezer for 12-18 h.
Note: It is recommended to stain and fix samples for mass cytometry on the same day rather than freezing, if possible.

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Note: The antibody dilutions vary based on the several factors such as the efficiency of the conjugation. Thus, it is optimal to always do the antibody titration when using a new batch of antibodies or using it on a different tissue type (See ''titration of antibodies and assessing signal spillover'' in the ''before you begin'' section).
Step 35 of ''Before you Begin'' and Figure 2 outline choosing the best dilution used for the rest of the protocol. Here, frozen samples are used to generate a single-cell suspension for staining. If the cells were not previously frozen, steps 3-5 should be skipped.

MATERIALS AND EQUIPMENT
1. Warm media and serum-free media to 37 C using a water bath.
Note: The media recipes are in the ''materials and equipment'' section.

Prepare a beaker with bleach for liquid waste.
Note: The amount of bleach should be enough so that the end volume has at least 10% bleach.
3. Label 15 mL conical tubes for the samples, which will be added. 4. Add 9 mL of media into the labeled 15 mL conical tubes. 5. Thaw the samples and quench the freezing media. a. Place the cryovials into the 37 C water bath for about a min or until about 80% thawed. b. Transfer the thawed cell suspensions into the labels 15 mL conical tubes.

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CRITICAL: Once thawed, frozen cell must be transfer quickly to media to minimize toxic exposure to DMSO.
6. Centrifuge the cell suspensions for 5 min at 300 3 g. 7. Discard the supernatant and resuspend in 1 mL of medium. 8. Count the cells using a hemacytometer. 9. Centrifuge for 5 min at 300 3 g and discard supernatant. 10. Resuspend the cells at 2 3 10 7 cells/mL of warm serum-free media.

Timing: 30 min
Cisplatin is used for identifying dead cells during data analysis. 11. Prepare 10 uM (23)   23. Incubate at 25 C for 10 min. 24. To prepare the surface antibody master mix, add the surface antibodies shown in Table 1 (in this case, all antibodies except anti-CD206 and anti-Gr-1) to Maxparâ Cell Staining Buffer to make 50 mL of antibody solution per sample.
Note: The volume of each antibody used for the master mix is based on the concentration determined by the titration (refer to the ''titration of antibodies and assessing signal spillover'' section).
Note: CD206 is stained during the intracellular staining process. Anti-Gr-1 recognizes both Ly6G and Ly6C at different binding sites than either clone of the Ly6G and Ly6C antibodies used in this panel. However, to avoid any potential steric hindrance, it is recommended to do sequential staining for anti-Ly6G and anti-Ly6C, followed by anti-Gr-1.

Fixing and intracellular antibody staining
Timing: 2 h The cells are fixed and permeabilized to allow for entry of the intracellular antigens staining antibodies, which then bind to and label the targeted intracellular antigens. 31. Gently vortex to resuspend the cell pellet in 1 mL of 13 Maxparâ Fix I Buffer. 32. Incubate for 30 min at 25 C to fix the cells. 33. During incubation, make 100 mL intercellular antibody master mix per sample by adding the intracellular antibodies to Maxparâ Perm-S Buffer. In this case, anti-CD206 is the only antibody used for intracellular labeling.
Note: The volume of each antibody used for the master mix is based on the concentration determined by the titration (refer to the ''titration of antibodies and assessing signal spillover'' section).
Note: If the intracellular target is a transcription factor or an intranuclear protein, it is recommended to perform the permeabilization using the Maxparâ Nuclear Antigen Staining Buffer Set (Fluidigm, Cat# 201063).
34. Wash the cells. a. Add 2 mL of Maxparâ Perm-S Buffer. b. Centrifuge at 800 3 g for 5 min. c. Discard supernatant. 35. Repeat the wash for a total of 2 washes. 36. Gently resuspend the pellet in 100 mL of intracellular antibody stain. 37. Incubate for 30 min at 25 C.

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38. Wash the cells two more times as done in step 34.

Fresh fix and DNA intercalator staining
Timing: 45 min, left overnight (12-18 h) In this step, the cells are stained with DNA intercalator, which allows for downstream identification of cell singlets. As DNA intercalator binds to nucleic acids, and cell-cell doublets have more DNA, doublets have a stronger DNA intercalator signal than singlets. Note: This solution can be prepared during the centrifugation in step 41 to save time.
44. Add 1 mL of the 13 intercalation solution and gently vortex to mix. 45. Incubate overnight (12-18 h) at 4 C to label the nucleated cells.
Pause point: The cells can be left up to 2 days at 4 C until acquisition.

Sample acquisition on CyTOFâ instrument
Timing: 1 day (3-4 h per sample) It is important to run the samples along with Fluidigm's EQä Four Element Calibration Beads, which allow for changes during sample acquisition to be monitored. The steps for sample acquisition and monitoring are as follow: 46. Wash the cells. a. Add 3 mL of Maxparâ Cell Staining Buffer. b. Centrifuge at 800 3 g for 5 min. c. Discard supernatant. 47. Resuspend the cells in 1 mL of Milli-Q water and count the cells. 48. Add an additional 3 mL to wash the cells and centrifuge at 800 3 g for 5 min. 49. Resuspend the pelleted cells with Milli-Q water containing 0.13 EQä Four Element Calibration Beads (diluted from 13 stock bottle) according to cell count to achieve 8-10 3 10 5 cells/mL. 50. Filter cells with a 40 mm cell strainer prior to introduction into the Heliosä, a CyTOFâ System. Note: Changes in sample acquisition can happen due to variations in instrument performance. Because of these variations in instrument performance, quality control of the instrument before and during sample acquisition is essential.
Note: This protocol focuses on the preparation and staining of cell suspensions along with the analysis of mass cytometry data. The process of acquiring the sample data is beyond the scope of this protocol. As such, samples should be submitted to a mass cytometry core specifically trained to run the samples for data acquisition.

Normalization
Timing: 1 h Data normalization is crucial to minimize technical variation. This process can be performed using the Fluidigm CyTOFâ software, which uses a standard ''Bead passport'' with standardized target values for the calibration beads. Using this tool, a bead's signal is detected and recorded throughout the sample acquisition, and the median intensity is calculated within a user-defined time interval. By assuming a linear rate of signal decay, the rate constant of signal decay in each interval is calculated against the global mean of signal intensity. Every event within the specified interval is multiplied by the rate constant. 51. Using the CyTOFâ software, select the raw FCS file, choose the ''Process'' tab, and select FCS processing. 52. Select the corresponding bead passport to the EQä Four Element Calibration Beads lot number used in data acquisition. 53. Select the time interval that the normalization factor will be applied to.
Note: By default, the software will use a 100 s interval.
Note: Another method of normalization was developed by the Nolan lab to mitigate the impact of signal drift on the data.

Concatenation (optional)
Timing: 1 h Sample acquisition may need to be run in various rounds because of the volume of the sample or a change in system performance during acquisition, such as a clog or unexpected shutdown. This results in multiple FCS files for a single sample which can then be concatenated into a single file. 54. In the CyTOFâ Software, select the FCS processing tab. Then select the raw FCS files to concatenate. 55. Click on the ''concatenate'' tab. The concatenated FCS file will be generated and ready for export.
CRITICAL: Make sure that the original data box is checked.
CRITICAL: Route the name and the directory of the new concatenated file. The normalization process will take place automatically once you concatenate.
Note: Instead of the CyTOFâ software, concatenation may be performed using Cytobank. However, data must be normalized before using this tool.

EXPECTED OUTCOMES
Due to the large number of antibodies used to run the mass cytometry panel, data analysis can be complex. This is particularly true in situations that require annotation of subpopulations that express a previously unidentified marker. However, mass cytometry analysis is facilitated by a number of publicly available analysis tools that rely mainly on dimensionality reduction. Several publications examine these tools in higher detail, which are not within the scope of this protocol (Kimball et al., 2018;Liu et al., 2019;Matos et al., 2017;Weber and Robinson, 2016). Here, we will discuss some commonly used advanced analytical tools available in Cytobank in the context of the questions they were designed to answer. We will also discuss the advantages and disadvantages of each analysis method and the ideal situation in which to run each one of them, which depends heavily on the biological question being asked.

QUANTIFICATION AND STATISTICAL ANALYSIS
A standard mass cytometry analysis workflow consists of the following steps: (1) Identification of different cell subsets using manual or automated gating detection, (2) Applying a clustering algorithm to detect the abundance of a specific cell subset and/or differential biomarker expression in cluster subsets across different conditions, and (3) visualization of cell clusters using dimensionality reduction.

Data visualization and gating
The first step of mass cytometry data analysis is performing data cleanup. This consists of the removal of unwanted events, dead cells, debris, normalization beads, true aggregates, and coincident ion clouds from raw data. The most common approach is to use the Gaussian distributionbased models available on Fluidigm Heliosä and CyTOFâ instruments. The four Gaussian parameters, residual, offset, center, and width, can be used to describe the shape of the distribution of pushes in a pulse, where a push is each instance of ions being sent to the detector and an individual pulse is composed of pushes integrated together to form an event (see Figure 4) (Lee and Rahman, 2019). These parameters are valuable for detecting well-formed pulses, eliminating coincident positive ion clouds, and to further exclude cell doublets. An example of using Gaussian-based parameter for single cell gating is illustrated in Figure 4.
To perform data cleanup: 1. Gate out the EQä Four Element Calibration Beads by plotting 140Ce against Time.
Note: 140Ce is tagged to the EQä Four Element Calibration Beads. 195Pt is from the Cell-IDä Cisplatin. 191Ir and 193Ir are from the Cell-IDä Intercalator.
2. Gate on the four Gaussian parameters sequentially (i.e., residual, center, offset, width) to eliminate unwanted non-Gaussian pulses as shown in Figure 4. 3. Select the ideal metal ion content and ion cloud size by plotting Event Length vs Time. 4. Gate on the live cells by plotting 195Pt vs time. 5. Remove doublets by applying two DNA gates (i.e.,191Ir,and 193Ir) vs Time. In both plots, the doublets will be the cloud with higher DNA content.

Automated gating
After data cleanup, the next step is to identify the different cell populations. This can be done using supervised or unsupervised gating to identify populations of interest. Below we will compare the advantages and the disadvantages of each method.

Unsupervised gating
This approach is done via clustering methods which place cell populations with similar phenotypes together in a low dimensional space. This allows the discovery of unknown cell populations from high dimension data analysis in a large dataset by comparing two groups (such as, control vs treated). A variety of advanced approaches are available to perform such analysis. For example, FlowSOM, SPADE, and Phonograph are tools used to perform high dimensional data analysis based on automated clustering. A variety of user-defined parameters, such as k-mean value, can determine the resolution and subculturing of immune cell populations. It is important to set these values to represent the expected subpopulations and avoid over clustering.

Supervised gating
This approach is useful when the experimental expectations and the cell type compositions in the samples are known. It is useful to discover and validate novel biomarkers by comparing two populations of interest in distinct groups through high-dimensional analysis. FlowDensity and OpenCyto are useful tools to perform automated gating using user-defined gates. The main advantage of using these tools is that the threshold for identifying the positive population is adjusted in a data-dependent manner for each sample rather than manually gating each sample, which can be subjective.

Cell subset detection (clustering)
Many clustering algorithms for mass cytometry have been published in recent years. However, the differences in their accuracy and sensitivity when identifying different cell populations is still a point of debate. An ideal algorithm for analyzing mass cytometry data should not only detect all major immune cell populations but also detects rare cell populations. Below, we discuss some commonly used clustering algorithms in Cytobank.
t-SNE: Clustering on dimensionality reduction channels (e.g., viSNE) is a more practical approach for samples consisting of heterogeneous cell subsets (high dimensional data). These include samples ll OPEN ACCESS containing cells with different and/or variable biomarker expression. In these situations, it is important to do t-SNE dimensionality reduction before applying any clustering algorithm such as SPADE or FlowSOM. However, because each dataset is different, it is difficult to determine when this approach is needed. Therefore, data must be explored in depth to determine the expression pattern and the heterogeneity of the sample. An example of using the t-SNE clustering approach to identify different immune cells infiltrating a GBM tumor microenvironment is shown in Figure 5.
Spanning-tree progression analysis for density-normalized events (SPADE): SPADE is one of the most widely used algorithms for immune cell mass cytometry data analysis. The main advantages of SPADE are that it overcomes computational challenges while lowering the risk of missing rare populations by performing density-dependent downsampling before hierarchical clustering. Density-dependent downsampling is an algorithm that extracts events to normalize the cell density. This allows for smaller cell populations not be overshadowed by larger cell populations. Utilizing hierarchical clustering enables SPADE to capture the continuity of phenotypes in the hematopoietic system.
FlowSOM: FlowSOM reveals how all markers behave on all cells by utilizing Self-Organizing Maps (SOMs) to shorten analysis time and improve clustering quality. The main advantage of this algorithm is that it is quick and eliminates the need for downsampling. In addition, it avoids the need to define the number of clusters in the output, which is a major drawback of SPADE which can introduce bias to the analysis.

LIMITATIONS
Be wary of environmental metal contamination. Reagents from other companies and certain dish cleaning detergents can add unwanted metal contamination. Additionally, this technique should be conducted in an area without traces of metals in the air, such as those generated when using flints and Bunsen burners.
This protocol has the preparation of the antibody mixes on the day of the main staining experiment.
To shorten the experiment time, the antibody mixes can be made the day prior and stored at 4 C in the dark.
Tissues can be harvested on the day of the main staining experiment to reduce loss of cell viability at the cost of a lengthier main experiment day.

TROUBLESHOOTING
Problem 1 When conjugating the purified antibodies to lanthanide metal labels, it is difficult to manage the timing of the different incubation periods due to only having one centrifuge, especially when the antibody reduction requires an exactly 30 min incubation (see step 2 in the ''conjugating the antibodies to metal isotopes'' section).

Potential solution
While the protocol may recommend that the polymer and antibody be processed at the same time, the polymer washes can be delayed until after the antibody reduction and purification are complete. The antibody reduction must be completed with the incubation timings given to prevent undesirable conjugations. On the other hand, the polymer wash can be delayed without consequence.

Problem 2
Since samples will be resuspended in water just prior to acquisition, cells are prone to degradation and debris formation (see step 49).

Potential solution
Proper fixation will protect cells from the hypotonic environment of the water before data acquisition.

Problem 3
Detector voltage may vary during sample acquisition, which may create high variability between samples (see step 50).

Potential solution
As part of the quality control for each sample, it is important to check the intensity of the beads in each sample. The best way to do that is by checking the median signal intensity of Eu153 on the beads and comparing it to the reference beads' intensity value. If the signal drops more than 25% in any sample, stop the acquisition, and recalibrate the detector voltage before running the sample again. Also, the coefficient of variance (CV) of the beads is reflective of the amount of error during ll OPEN ACCESS data acquisition. High CVs are indicative of a reduced ability to accurately detect minor changes in protein expression levels between cells.

Problem 4
During sample acquisition, oxidation of metal conjugated antibodies may occur, resulting in a 16 dalton (Da) increase in the mass channel of that isotope (for example Nd 146 oxides will be recorded as Dy162). This can be a source of cross reactivity between the metal isotopes and should be considered during the panel design (see step 50).

Potential solution
EQä Four Element Calibration Beads can be used to monitor alteration due to oxide formation during sample acquisition by calculating the ratio between the dual counts of Ce140 and Gd156. Also, Fluidigm recommends tuning the instrument using CyTOFâ Tuning Solution (Fluidigm, SKU, 201072) to a ratio threshold of 3%. Due to the trade-off nature between oxidation and signal intensity, increasing signal intensity during tuning typically indicates a higher oxidation ratio.

Problem 5
Cell signal intensity may decrease during acquisition because of instrument performance or sample degradation.

Potential solution
While instrument performance can be tracked using EQä Four Element Calibration Beads, cell-specific degradation cannot. First, identify the Ce140+ DNAÀ EQä Four Element Calibration Beads population and the Ce140ÀDNA+ cell population. Next, plot time vs. a measured parameter (e.g., Eu153 for beads or CD45 for cells) and calculate the slope for each. A slight negative slope is generally expected, reflecting gradual loss of instrument sensitivity over time. A steeper slope for both cells and beads indicates a more rapid loss of instrument sensitivity over the period of acquisition, while a steeper slope for measured cell parameters relative to the measured bead parameters can indicate sample degradation. This sample degradation is indicative of poor sample fixation.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Maria G. Castro, mariacas@med.umich.edu.

Materials availability
This study involved tumor tissue from SB glioma mouse models. The protocol for generation of this model is as described in Garcia-Fabiani et al. (2020).

Data and code availability
No datasets or code was generated or analyzed during this study.