May 13, 2026
Murine CyTOF Panel Staining and Fixation - University of Minnesota TMC
- 1University of Minnesota
- Cellular Senescence Network (SenNet) Method Development Community
Protocol Citation: Patrick Fehrenbach 2026. Murine CyTOF Panel Staining and Fixation - University of Minnesota TMC. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7oo43vwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 12, 2026
Last Modified: May 13, 2026
Protocol Integer ID: 316907
Keywords: CyTOF, SenNet, cytof acquisition of murine cell suspension, murine cytof panel staining, murine cell suspension, cytof acquisition, cytometry fluidic, cytof, cellular proteomic profile, cellular proteomic profiles of dozen, murine, conjugated antibody, mass spectrometry
Funders Acknowledgements:
National Institutes of Health
Grant ID: U54AG079754
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Abstract
This protocol outlines permeabilization, staining, fixation, and CyTOF acquisition of murine cell suspensions. CyTOF leverages heavy metal-conjugated antibodies; cytometry fluidics; and mass spectrometry to build cellular proteomic profiles of dozens of targets simultaneously.
Materials
Cell media (RPMI + 10% FBS + Pen/Strep)
Benzonase (if using frozen samples)
Maxpar® Water (500 mL – Product Number – 201069, Standard Biotools)
10X PBS pH 7.2 (Rockland 10X PBS, Code Number: MB-008, Lot Number: 33834, Size: 1L)
CyPBS (Dilute 10X Rockland PBS 1:10 in Maxpar water or Maxpar® PBS – Product number – 201058, Standard Biotools)
Maxpar® Cell Staining Buffer (CSB)—500 mL (Fluidigm; Part No. 201068)
eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set (Catalog number: 00-5523-00)
1. Fixation/Permeabilization Concentrate (cat. 00-5123)
2. Fixation/Permeabilization Diluent (cat. 00-5223)
3. Permeabilization Buffer (10X) (cat. 00-8333)
Fc Receptor Blockade (TruStain FcX™ PLUS, anti-mouse CD16/32, Biolegend 156604)
EMD Millipore Ultrafree™-MC Centrifugal Filter Devices with Durapore™ Membrane (Fisher – Product No. UFC30VV00)
Cisplatin solution (Cell-ID™ Cisplatin—100 μL; Fluidigm; Part No. 201064)
Fixation solution (16% PFA, EM Sciences)
Intercalation solution (Cell-ID™ Intercalator-Ir—125 μM; Fluidigm; Part No. 201192A)
Maxpar Cell Acquistion Solution (CAS)–100 mL (Fluidigm; Part No. 201237)
Eppendorf 5418 for 500 x g RT prior to fixation and @ 800 x g RT post-fixation
Filtered pipette tips
35um blue cap FACS tube (Falcon; 5mL polystyrene round-bottom tube with cell-strainer cap; Reference No. 352235)
Safety warnings
Caution: This solution contains formaldehyde, which is toxic and a suspected carcinogen. Contact with eyes, skin and mucous membranes should be avoided. Wear proper protective clothing and gloves.
Use filtered pipette tips only!
Ethics statement
All murine experiments have been reviewed and approved by the Institutional Animal Care and Use Committee at the University of Minnesota.
Reagents
Dilute 10X Rockland PBS 1:10 in Maxpar water to prepare CyPBS. Alternatively, use Maxpar® PBS (Product number – 201058, Standard Biotools). Store at 4°C.
eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set:
Fixation/Permeabilization Concentrate (cat. 00-5123): 30 mL. Store at 2-8°C. Avoid agitation. Use within 6 months of opening. This is a 4X stock solution that must be diluted prior to use with the Fixation/Permeabilization Diluent. Dilute 1 part Concentrate with 3 parts Diluent. Caution: This solution contains formaldehyde, which is toxic and a suspected carcinogen. Contact with eyes, skin and mucous membranes should be avoided. Wear proper protective clothing and gloves.
Fixation/Permeabilization Diluent (cat. 00-5223): 100 mL. Store at 4°C. The diluent is intended to be used in combination with the Fixation/Permeabilization Concentrate.
Permeabilization Buffer (10X) (cat. 00-8333): 100 mL. Store at 2-8°C. Prior to use, this should be diluted 10-fold in distilled water. Note: The 10X Permeabilization Buffer has a natural tendency to precipitate, however, its function is not affected by this. To clarify, the solution can be filtered after dilution to a 1X working solution.
Prepare Fc Receptor Blockade by using TruStain FcX™ PLUS (anti-mouse CD16/32) Antibody (Biolegend 156604) at 0.25 μg per 10^6 cells.
Make 0.5 μM cisplatin solution by diluting 5 mM cisplatin (Cell-ID™ Cisplatin—100 μL; Fluidigm; Part No. 201064) stock 1:10000 in CyPBS. Dilute 0.13 μL cisplatin stock in 1.3 mL CyPBS.
Make 2X (4.0% HCHO) fixation solution by diluting 16% PFA (EM Sciences) 1:4 in CyPBS. Dilute 1.0 mL 16% PFA in 3.0 mL CyPBS.
Prepare 62.5 nM intercalator solution by diluting 125 μM intercalator stock (Cell-ID™ Intercalator-Ir—125 μM; Fluidigm; Part No. 201192A) 1:2,000 in Maxpar Fix/Perm Buffer—250 mL (Fluidigm; Part No. 201067). Dilute 1.0 μL intercalator in 10 mL Maxpar Fix/Perm Buffer.
Can use Eppendorf 5418 for 500 x g RT prior to fixation and @ 800 x g RT post-fixation. Use filtered pipette tips only!
Cisplatin labeling
Centrifuge sample tubes at 500 xg for 5 min. at 4°C.
Incubate at 4°C for 5 min.
Add 1 mL CSB to each tube. Spin at 500 xg for 5 min. at 4°C.
Aspirate supernatant and resuspend in 1.5 mL CSB.
Spin down at 500 xg for 5 min. at 4°C.
Fc Block
Aspirate supernatant and resuspend in 50 μL CSB with Fc Block [Master Mix – (N x 3 μL Fc Block) + (N x 47μL CSB)]. Mix well. N=Total number of samples
Incubate at 4°C for 10 min.
Surface antibody staining
Add 50 μL antibody cocktail to each tube. Resuspend and incubate with gentle agitation at 4°C for 30 min.
Add 1.5 mL CSB to each sample and spin down at 500 xg for 5 min. at 4°C.
Aspirate supernatant and resuspend in 1 mL CSB. Spin down at 500 xg for 5 min. at 4°C.
Stain Cells with Nuclear/Intracellular Antibodies (eBiosciences reagents)
Aspirate CSB and add 1 mL of Foxp3 Fixation/Permeabilization working solution to each tube and resuspend.
Incubate for 30 minutes at 2-8°C. Protect from light. (Mouse samples can be incubated for up to 18 hours at 2-8°C in the dark).
Add 2 mL of 1X Permeabilization Buffer to each tube and centrifuge samples at 800 x g for 5 minutes at 4°C. Discard the supernatant.
Resuspend pellet in 50 μL of 1X Permeabilization Buffer.
Add 50 μL of Intracellular Antibody Master Mix to cells and incubate for 45 minutes at 4°C.
Add 2 mL of 1X Permeabilization Buffer to each tube and centrifuge samples at 800 x g for 5 minutes at 4°C. Discard the supernatant.
Repeat Step 23.
Fixation
Resuspend in 500 μL CyPBS. Add 500 μL 2X fixation solution to each tube. Vortex and incubate at 4°C ON or for 30 minutes with gentle agitation.
After fixation, add 1 mL CSB and spin down at 800 xg for 10 min at 4°C.
DNA intercalation
Aspirate supernatant and resuspend in 1 mL Intercalation solution. Mix well and incubate with gentle agitation in 4°C overnight or for 30 minutes.
Spin down at 800 xg for 10 min at 4°C.
Aspirate supernatant and resuspend in 2 mL CyPBS. Spin down at 800 xg for 10 min. at 4°C.
Resuspend in 1 mL of EQ Beads/ HPLC grade water (1:10) and count cells.
Prior to acquisition dilute samples to approximately 5x105 cells/mL. Thoroughly resuspend EQ beads by shaking vigorously for at least 1 minute.
Acquisition
Filter through 35um blue cap FACS tube (Falcon; 5mL polystyrene round-bottom tube with cell-strainer cap; Reference No. 352235).
Inject 500uL of filtered sample into sample port; repeat for all samples, washing sample loop between samples with HPLC-grade water
For issues with machine clogging, consult CyTOF2 user manual to help isolate location of problem.
CyTOF_2_ug_C7-UM13-01_v5.pdf9.2MB Protocol references
"Fluidigm CyTOF2 Mass Cytometer User Manual" Standard Biotools Protocol Document PN 400200 A5.