Oct 15, 2025
JAX-Sen: Single Cell Dissociation of Mouse Placenta
- Ramalakshmi Ramasamy1,
- Riley Sheldon1,
- Cole Lorig1,
- Jessica Garofalo1,
- Patrick Fleming1,
- Juliana Alcoforado Diniz1,
- Eric Loucks1,
- Paul Robson1,2
- 1The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA;
- 2Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, Farmington, CT, USA
- Cellular Senescence Network (SenNet) Method Development Community
Protocol Citation: Ramalakshmi Ramasamy, Riley Sheldon, Cole Lorig, Jessica Garofalo, Patrick Fleming, Juliana Alcoforado Diniz, Eric Loucks, Paul Robson 2025. JAX-Sen: Single Cell Dissociation of Mouse Placenta. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl46dm8go5/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: October 15, 2025
Last Modified: October 15, 2025
Protocol Integer ID: 229817
Keywords: JAX-Sen, single cell dissociation of mouse placenta, whole mouse placenta, mouse placenta, mouse placenta this protocol, submission for single cell rna, single cell rna, placenta, single cell dissociation, mouse strain, single cell suspension, female fetus, cell, live cell, enzymatic digestion, cell rna, rna
Funders Acknowledgements:
National Institute on Aging (NIA) JAX-Sen Senescence Tissue Mapping Center
Grant ID: U54 AG079753
Abstract
This protocol outlines the process of acquiring a single cell suspension prior to submission for single cell RNA sequencing. Whole mouse placentas were collected from male and female fetuses across 8 mouse strains (WSB, A/J, NOD, NZO, 129S1, PWK, CAST, C57Bl/6). Placentas were collected at 4 different timepoints for the C57Bl/6 strain (E9.5, E11.5, E13.5, and E17.5) and 1 timepoint (E13.5) for the other 7 strains. The whole mouse placenta is minced manually, undergoes two enzymatic digestions and ACK Lysis, and gets carefully filtered. Cells are then sorted to enrich for live cells and eliminate cellular debris.
Troubleshooting
Reagents and Materials
Day before:
- Scissors and tweezers (cleaned with ethanol)
- Autoclave bags and tape
- Autoclave tweezers and scissors
Day-of:
- Ice bucket and ice
- TrypLE
- Dispase
- RNase inhibitor
- DNase I
- Collagenase IV
- 1X PBS
- DMEM
- FBS
- EDTA
- BSA
- 15mL conical tubes
- 50mL conical tubes
Equipment:
- Centrifuge
- Benchtop centrifuge
- Rotating Incubator
- LUNA cell counter
Cool down centrifuge and benchtop centrifuge to 4oC
Heat up rotator to 37oC for Digest 2 (warm digest)
Fill ice bucket
Prepare reagents, listed below
Digest 1 (enough for 4 samples)
- 1937.5uL TrypLE
- 50uL Dispase
- 12.5uL RNase inhibitor
- 25uL DNase I
Digest 2 (enough for 5 samples)
- 1.25mg collagenase IV
- stock is 2mg/mL; need 0.625mL for one Digest 2
- 12.5uL RNase inhibitor
- Bring to 2.5mL with PBS
Ice-cold PBS
- 50mL PBS
Stop buffer (DMEM with 10% FBS)
- 45mL DMEM
- 5mL FBS
CS buffer (need 6mL per sample, make according to orange chart)
- EDTA
- PBS
- 10% BSA
- FBS
| A | B | C | D | E | F | G | H | I | |
| Stock conc. | Working conc. | 50 ml | 100 ml | 250 ml | 500 ml | 20 ml | |||
| EDTA | 0.5 M | 2 mM | 0.2 | 0.4 | 1 | 2 | 0.08 | ml | |
| BSA | 10% | 2% | 10 | 20 | 50 | 100 | 4 | ml | |
| FBS | 100% | 15% | 7.5 | 15 | 37.5 | 75 | 3 | ml | |
| DPBS | 1X | make up | 32.2 | 64.4 | 161 | 322 | 12.88 | ml |
*Keep samples/cells cold on ice throughout entire protocol
Placenta Dissociation:
- Ice bucket and ice
- Autoclaved tweezers and scissors
- Placenta samples & buffer (keep on ice)
- 5mL eppendorfs
- Ice-cold PBS (keep on ice)
- STOP Buffer (keep on ice)
- Digest 1 (keep on ice)
- 70um cell strainers
- 15mL conical tubes
- 2mL eppendorfs
- Digest 2 (keep on ice until otherwise indicated)
- ACK Lysing Buffer
- CS Buffer (keep on ice)
- Flowmi 40um cell strainers
- New 2mL tubes
Cell Counting with LUNA
- AOPI
- LUNA chip
Cell Staining/FACS:
- Ice bucket and ice
- Cell staining buffer
- Calcein
- PI
- FACS tubes
- 5mL eppendorfs
- 0.04% BSA
- Aluminum foil
Dissociation Protocol:
Remove placenta from original tube with tweezers and place into labeled 5mL tube on ice
Add 1mL of the MACS buffer from each sample’s original tube
Mince with scissors on ice
Add 2mL cold PBS; Coat wide P1000 with STOP buffer and pipette gently up and down to mix
Centrifuge at 800 x g, 4oC for 5 minutes
Aspirate supernatant and resuspend pellet in 2mL ice-cold PBS, pipette with coated wide P1000 to mix
Centrifuge at 800 x g, 4oC for 5 minutes
Aspirate supernatant and resuspend pellet in 2mL ice-cold PBS, pipette with coated wide P1000 to mix
Centrifuge at 800 x g, 4oC for 5 minutes
Aspirate supernatant
Add 500uL of DIGEST 1, pipette up and down with coated wide P1000 after adding
Warm up DIGEST 2. Take samples in DIGEST 1 to cold room to rock; coat wide P1000 with STOP buffer and mix samples for 25 minutes
a. While DIGEST 1 runs, put DIGEST 2 in 37oC incubator to warm up & prep tubes + 70um filters
Stop first digestion by adding 500uL STOP buffer
Filter cells through 70um cell strainers
a. First, retrieve 15mL tubes and 70um stainers for every sample. Label the 15mL tubes and strainer. Coat the strainers with 1000uL STOP buffer.
b. Also, label a 2mL tube for each sample
Wash 1mL of STOP buffer through the strainer after filtering cells
Collect the retentate on the strainer with wide P1000 and transfer to the new labeled 2mL tube
Add 500uL DIGEST 2 to the 2mL tube, pipette up and down with coated wide P1000 after adding
Put tubes in 37oC rotator for 10 minutes
a. Halfway through, pipette up and down with coated wide P1000
Stop second digestion by adding 500uL STOP buffer, mix up and down
Filter contents of the 2mL tube through the same strainer as before
Wash 1mL STOP buffer through the filter
Centrifuge the 15mL tubes at 800 x g, 4oC for 5 minutes to pellet cells
Aspirate supernatant and be careful not to disturb the pellet
Resuspend pellet in 2mL ACK Lysing buffer, mix up and down
Incubate on ice for 5 minutes
Add 5mL CS buffer to stop ACK lysis
Collect cells by centrifugation at 800 x g, 4oC for 5 minutes
Resuspend pellet in 1000uL CS buffer, making sure the pellet is disrupted appropriately
Use P1000 to collect the cell suspension, then slowly pipette out through a Flowmi 40um cell strainer into a new 2mL tube
Cell Counting with LUNA:
Mix a 1:1 ratio of cell suspension and AOPI
a. 10uL cells and 10uL AOPI
Load 10uL of mixture into LUNA chip
Count with LUNA
a. Write down: Number of cells (cells/mL), viability (%), cell size (um)
After counting with AOPI, spin 2mL tubes down in centrifuge on bench to collect cell pellet
a. 800 x g, 5 minutes, 4oC
Aspirate supernatant carefully with P1000 set to 1000uL and resuspend according to Cell Staining/FACS protocol below
Cell Staining/FACS:
Resuspend pellet in 300uL cell staining buffer
a. Resuspend pellet with highest cell count/viability combination in 350uL cell staining buffer
Transfer 50uL from the 350uL resuspension into separate FACS tube (to serve as unstained control)
a. Add 200uL cell stain buffer for total volume of 250uL
Stain all other samples with Calcein and PI
a. Live cells, Calcein: 10uL for every 300uL sample
b. Dead cells, PI: 1uL for every 100uL sample
i. 300uL sample totals: 10uL calcein and 3uL PI
c. Add on ice and cover with tinfoil (stains are light sensitive), don't mix!
d. Write down time of staining on foil covering ice bucket
After staining with both Calcein and PI, pipette up and down to mix, then add to FACS tube
a. Pipette straight down and directly onto the filter
Create collection tubes for FACS
a. Add 100uL 0.04% BSA in PBS into a labeled 5mL microcentrifuge tube
Sort for live cells and submit for scRNAseq