May 14, 2026
Murine Tissue Digestion Protocol for CyTOF - University of Minnesota TMC
- 1University of Minnesota
- Cellular Senescence Network (SenNet) Method Development Community
Protocol Citation: Patrick Fehrenbach 2026. Murine Tissue Digestion Protocol for CyTOF - University of Minnesota TMC. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8449dg2w/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 13, 2026
Last Modified: May 14, 2026
Protocol Integer ID: 316904
Keywords: CyTOF, SenNet, Liver, Adipose, Brain, Mouse, murine tissue digestion protocol for cytof, murine tissue digestion protocol, downstream analysis by cytof, cytof, types of murine, origin tissue, protocol details the step, tissue, murine, liver, protocol detail
Funders Acknowledgements:
National Institutes of Health
Grant ID: U54AG079754
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Abstract
This protocol details the steps used to prepare three types of murine-origin tissues — adipose, brain, and liver — for downstream analysis by CyTOF.
Adipose Tissue
CyTOF collagenase protocol 2024-5
1. Collect fat in 5 ml RP10 (RPMI+pen/strep+10%FBS)
2. Rinse in PBS, pat dry on paper towel
3. Mince well
4. Collagenase digest
Collagenase Media
| A | B | C | D | E | |
| Collagenase Media | stock | final | 10ml/mouse | units | |
| 10xHBSS | 10X | 1X | 1 | mL | |
| Calcium chloride | 1 M | 1.2 mM | 10 | uL | |
| Magnesium chloride | 1 M | 1 mM | 12 | uL | |
| Zinc Chloride | 0.1M | 0.8 mM | 80 | uL | |
| water | 9.40 | mL | |||
| BSA | 3% | .3 | g | ||
| collagenase II | 0.8 mg/ml | 8 | mg |
final 10ml/mouse
5. Incubate 37°C, shaking 200 rpm 10-30 minutes- check every 10 minutes
6. Filter 100 um Have extra RP10 to help filter
7. Centrifuge 5 minutes 800 xg; 4°C
8. (IF want for other experiments: Remove adipocytes; add Trizol)
9. Resuspend SVF pellet in 5 ml RP10
10. Centrifuge 5 minutes 800 xg; 4°C; discard supernatant
11. Lyse RBC with 1 ml ACK solution; Room temp 2 min
12. Add 4 mls RP10
13. Filter 40 um Have extra RP10 to help filter
14. Centrifuge 5 minutes 800 xg; 4°C
15. Resuspend in 1 ml RP10 Gently resuspend cells
Keep on ice
Brain tissue
NOTE: Miltenyi Biotec Isolation and cultivation of neurons from adult mouse brain protocol
2. Protocol
2.1 Preparation of brain dissociation
▲ For subsequent cell separation and cultivation it is recommended
to dissociate at least 800 mg of adult mouse brain tissue.
▲ Volumes given below are for one adult mouse brain (max.
500 mg) in 1980 μL enzyme mix. When working with less than
500 mg, use the same volumes as indicated. When working with
higher tissue quantitities scale up all reagent volumes and total
volumes accordingly.
▲ A swinging bucket rotor is recommended for centrifugation,
e. g., Heraeus‱ Multifuge 4KR by Thermo Fisher‱ Scientific.
-
Enzyme P is ready to use. Prepare aliquots of appropriate
volume to avoid repeated freeze-thaw-cycles. Store
aliquots at –20 °C. This solution is stable for 6 months.
Resuspend the lyophilized powder in the vial labeled Enzyme
A with 1 mL Buffer A. Do not vortex. This solution should then
be aliquoted and stored at –20 °C for later use. Avoid repeated
freeze-thaw-cycles.
-
Prepare enzyme mix 1 and enzyme mix 2 according to the
table below.
Enzyme mix 1 Enzyme mix 2
Enzyme P
50 µL
Buffer Z
1900 µL
Buffer Y
20 µL
Enzyme A
10 µL
Preparation of 1× Red Blood Cell Removal Solution
-
Dilute the Red Blood Cell Removal Solution (10×) 1:10 with
double-distilled water (ddH₂O), for example, dilute 0.1 mL of
cold Red Blood Cell Removal Solution (10×) with 0.9 mL cold
ddH₂O.
▲ Note: Do not use deionized water for dilution!
-
Store the prepared 1× Red Blood Cell Removal Solution at
2–8 °C. Discard unused solution at the end of the day.
Preparation of cell culture dish
-
Prepare the following medium: MACS Neuro Medium
containing 2% MACS Neuro Brew-21, 1% Penicillin/
Streptomycin and 0.5 mM L-Glutamine.
-
-
Coat the culture dish (24-well plate) with 0.01% Poly-L-
Lysine overnight at 37 °C and wash three times with ddH₂O
afterwards. Let the culture dish dry under sterile conditions.
2.1.1 Dissociation protocol
▲ For details on the use of the gentleMACS‱ Octo Dissociator with
Heaters, refer to the user manual.
▲ A maximum of one mouse brain (max. 500 mg) in 2 mL enzyme
mix can be processed in one C Tube.
▲ For cell culture experiments subsequent to tissue dissociation,
all steps should be performed under sterile conditions.
1. Remove the mouse brain. Wash the brain in cold D-PBS.
2. Prepare the appropriate volume of enzyme mix 1 (refer to table
in chapter 2.1) and transfer it into a gentleMACS C Tube.
3. Place the brain on a petri dish and cut it into 8 sagittal slices
using a scalpel.
4. Transfer the tissue pieces into the C Tube containing 1950 μL
of enzyme mix 1.
5. Transfer 30 μL of enzyme mix 2 into the C Tube.
6. Tightly close C Tube and attach it upside down onto the sleeve
of the gentleMACS Octo Dissociator with Heaters.
7. Run the gentleMACS Program 37C_ABDK_01.
8. After termination of the program, detach C Tube from the
gentleMACS Octo Dissociator with Heaters.
9. (Optional) Centrifuge briefly to collect the sample at the
bottom of the tube.
10. Resuspend sample and apply it to a MACS SmartStrainer
(70 μm) placed on a 50 mL tube.
▲
▲ Note: Moisten MACS SmartStrainer with buffer before use.
▲
▲ Note: When upscaling the reagent volume and total volumes, increase also
the number of MACS SmartStrainers (70 μm). One MACS SmartStrainer
(70 μm) can be used for one adult mouse brain.
▲
▲ Note: Dissociated tissue can be removed from the closed C Tube by pipetting
through the septum-sealed opening in the center of the cap of the C Tube. Use
ART 1000 REACH 1000 μL pipette tips.
▲
▲ Note: Cells with a diameter >70 μm may be lost. To obtain these cells within
the flow through, use a cell strainer with an appropriate mesh size.
11. Apply 10 mL of cold (4 °C) D-PBS onto the MACS SmartStrainer
(70 μm).
12. Discard MACS SmartStrainer (70 μm) and centrifuge
cell suspension at 300×g for 10 minutes at 4 °C. Aspirate
supernatant completely.
13. Proceed to 2.1.2 for debris and red blood cell removal.
2.1.2 Debris and red blood cell removal
▲ Volumes given below are for the cell suspension from up to two
adult mouse brains. When working with higher tissue quantities,
scale up all reagent volumes accordingly.
▲ A maximum of cell suspension from two adult mouse brains can
be processed in one 15 mL reagent tube.
▲ Always use pre-cooled buffers and solutions (4 °C).
Debris Removal Solution D-PBS Overlay
(D-PBS)
1 brain
(400–500 mg)
900 µL 3100 µL 4 mL
2 brains
(800–1000 mg)
1800 µL 6200 µL 4 mL
1. Resuspend cell pellet carefully with the appropriate volume
of cold D-PBS according to the table above and transfer cell
suspension to a 15 mL tube. Do not vortex.
2. Add appropriate volume of cold Debris Removal Solution.
3. Mix well.
4. Overlay very gently with 4 mL of cold D-PBS.
▲ ▲ Note: Pipette very slowly to ensure that the D-PBS phase overlays the cell
suspension and phases are not mixed.
5. Centrifuge at 4 °C and 3000×g for 10 minutes with full
acceleration and full brake.
▲ ▲ Note: If centrifuges give suboptimal centrifugation, the acceleration and
brake can be reduced.
6. Three phases are formed. Aspirate the two top phases
completely and discard them.
7. Fill up with cold D-PBS to a final volume of 15 mL.
8. Gently invert the tube three times. Do not vortex!
9. Centrifuge at 4 °C and 1000×g for 10 minutes with full
acceleration and full brake. Aspirate supernatant completely.
10. Resuspend cell pellet from up to two adult mouse brains
carefully in 1 mL of cold 1× Red Blood Cell Removal Solution.
Do not vortex.
11. Incubate for 10 minutes in the refrigerator (2−8 °C).
12. Add 10 mL of cold D-PBS/BSA buffer.
13. Centrifuge at 4 °C and 300×g for 10 minutes. Aspirate
supernatant completely.
Liver tissue
Protocol of preparation of single cells suspensions from mouse liver
1. Euthanized mice and collect liver.Remove gall bladder from the liver with forceps and preserved the liver tissues in a cold MACS tissue storage solution supplemented with Reagent E (1:200), taken from the Liver Perfusion Kit, mouse and rat (130-128-030) on ice. The liver tissue can be stored up to 2 hours.
2. Liver dissociation protocol
1. Prepare dissociation mix by pipetting 4.7mL DMEM into a gentle MACS TMC Tube. Add 200 μL Enzyme D solution, 100 μL Enzyme R solution, and 20 μL Enzyme A solution.
2. Incubate dissociation mix for 30 minutes at 37°C in an incubator or 15 minutes in a water bath.
3. Rinse liver with DMEM.
4. Transfer liver into the CTube containing the dissociation mix.
5. Tightly close CTube and attach it upside down onto the sleeve of the gentleMACS Dissociator.
▲ Note: Close C Tube tightly beyond the first resistance.
▲ Note: It has to be ensured that the sample material is located in the area of the rotor/stator.
6. Run the gentle MACS Program m_liver_03
7. After termination of the program, detach CTube from the gentleMACS Dissociator.
8. Incubate sample for 30 minutes a t37°C under continuous rotation
9. Attach CTube upside down onto the sleeve of the gentleMACS Dissociator.
10. Run the gentleMACS Program m_liver_04.
11. After termination of the program, detach C Tube from the gentleMACS Dissociator.
12. Resuspend sample and apply the cell suspension to a MACS‱ SmartStrainer (100 μm).
13. Wash the filter with 5 mL DMEM with stable glutamine.
▲ Note: For maximum cell recovery, rinse the used C Tube with wash buaer before transfer to filter.14. Discard the filter and centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely. _____________________________________________________________________
3. Add 1-2 mL of ACK lysis buffer(ThermoFisher-A1049201)and incubate the cells in 5- 10 minutes at RT.
4. Resuspend the cells in 10mL 1XDPBS. Centrifuge cell suspension at 300×g for 7 minutes.
5. Remove the supernatant and resuspend cells with appropriate amount of PEB buffer (Dilute MACS BSA Stock Solution (# 130-091-376) 1:20 with autoMACS‱ Rinsing Solution(# 130-091-222). Keep buffer cold.)
6. Count the cells stained with trypan blue using hemocytometer
7. Adjust the volume to 2 million cells per 1mL in PEB buffer.
Protocol
CyTOF_tissue_digestion_rprotocols_Adipose_brain_liver.pdf326.6KB Protocol references
"Isolation and cultivation of neurons from adult mouse brain." Miltenyi Biotec, Protocol Document SP0093.05.
"Liver dissociation kit." Miltenyi Biotec, Protocol Document 140-004-706.04.