Method Article
Mouse fetal liver erythroid population separation using FACS.
https://doi.org/10.21203/rs.3.pex-1246/v1
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Through-out erythropoiesis cells undergo several morphological and regulatory changes. In mouse, these changes can be characterized through isolation of specific intermediate populations based on the surface levels of Ter119 (Glycophorin A-associated protein/Ly-76) and CD71 (Transferrin receptor 1)1-2. This method takes roughly one day and can be used to generate sufficient cells for most high-throughput sequencing techniques with low input adaptations.
- PBS (Invitrogen: 10010031)
- Bovine Serum Albumin, BSA (Sigma: A4503)
- Glucose (Sigma: G7021)
- EDTA (Invitrogen: 15575-038)
- RPMI (ThermoFisher Scientific: 21875034)
- Fetal Calf Serum, FCS (Sigma: F7524)
- DNaseI (VWR, A3778.0050)
- MgCl2 (Ambion: AM9530G)
- e13.5 Mouse fetal livers 12-20 embryos
- Streptavidin MicroBeads (Miltenyi: 130-048-101)
- Hoechst 33258 (ThermoFisher Scientific: H3569)
Antibodies
- ChromePure Rabbit IgG, 20mg/ml (Jackson, 015-000-003)
- CD41-FITC (BD Bioscience, Clone: MWReg30, Cat: 553848)
- CD45R/B220-FITC (BDBioscience, Clone: RA3-6B2, Cat: 553087)
- CD3e-FITC (BDBioscience, Clone: 145-2C11 553061, Cat: 6053673)
- CD11b/Mac-1-FITC (BDBioscience, Clone: M1/70, Cat: 557396)
- Ly-6G and Ly-6C (Gr-1)-FITC (BDBioscience, Clone: RB6-8C5, Cat: 553126)
- Ter119-Biotin (BD Bioscience, Cat: 553672)
- Streptavidin-APC (Biolegend, Cat:405207)
- CD71-PE-Cy7 (Biolegend Clone: RI7217, Cat: 113811)
DNase stock solution:
- 1x PBS
- 5 mg/mL DNase I
- 100mM MgCl2
Staining solution
- 1x PBS
- 0.2% BSA
- 5 mM glucose
* For liver dissociation add 1 mL DNase stock solution to 50 mL.
* For sorting include 5mM EDTA.
Separation buffer
- PBS (pH 7.2)
- 0.5% BSA
- 2 mM EDTA
Collection buffer
- RPMI
- 10% FCS
- Partec CellTrics 30 µm cell filters (Sysmex: 04-004-2326)
- NucleoCounter NC-3000 cell counter, or equivalent (Chemometec)
- LS separator columns (Miltenyi: 130-042-401)
- Pre-sepration filter (30 µM) (Miltenyi:130-041-407)
- MACS MultiStand, or equivalent (Miltenyi: 130-042-303)
- QuadroMACS Separator, or equivalent (Miltenyi: 130-090-976)
- BD FACSAria™ Fusion Sorter, with 100 μM nozzle, or equivalent (BD Bioscience)
Dissociation of mouse fetal livers.
** Chill centrifuge to 4ºC **
** Add 1 mL DNaseI stock solution to 50mL of ice-cold Staining Solution. **
1. Collect livers from e13.5 mouse embryos (12-20 required).
2. Dissociate fetal liver tissue into 3 mL ice-cold staining solution by gently pipetting up and down with a P1000 pipette.
3. Transfer cells to a 12x75 mm tube.
4. Pellet cells by centrifugation at 200 rcf for 5 min (4ºC).
5. Discard supernatant and gently resuspend in 1 mL of ice-cold staining solution to wash cells.
6. Pass resuspended cells through a 30 µm filter.
7. Rinse filter with 2 mL ice cold staining solution ot dislodge cells stuck to the membrane.
8. Pellet cells by centrifugation at 200 rcf for 5 min (4ºC).
9. Discard supernatant and gently resuspend in 1 mL of ice-cold staining solution.
10. Count cells (for NC-3000 cell counter use Solution 13).
Mature erythoid cell depletion.
1. Adjust volume of cells in staining solution to achieve 0.5-1 x 107 cells per 100 μL.
2. Block Fc receptors by adding ChromePure Rabbit IgG (final concentraion 200 µg/mL).
3. Add 10 µL of Ter119-biotin per mL of cells.
4. Incubate on ice for 30 min.
5. Wash the cells by adding 3 mL of cold staining solution.
6. Pellet cells by centrifugation at 200 rcf for 5 min (4ºC).
7. Resuspend cells in 90 μL cold staining buffer per 107 cells.
8. Set aside 2 x 106 cells (18 µL) for staining controls and make up to 1 mL with staining solution. Keep on ice.
9. Set aside 8 x 106 cells (72 µL) for unenriched sort and make up to 100 µL with staining solution. Keep on ice.
10. Add Streptavidin MicroBeads to the remaining cells (10 μL per 107 cells).
11. Mix well by pipetting and incubate for 15 min at 4°C.
12. Wash the cells by adding 3 mL of separation buffer.
13. Pellet cells by centrifugation at 200 rcf for 5 min (4ºC).
14. Whilst cells are washing, place LS separator columns (1 for every 1x108 cells) into
the magnet and rinse with 2 mL of separation buffer.
15. Discard supernatant and resuspend pellet in 500 μL of separation buffer per 108 cells.
16. Place an 30 µm pre-separation filter in each LS column, and a 15 mL collection tube below the column.
17. Apply 500 µL of resuspended cells to each LS column, collecting the flow through which contains Ter119-negative progenitor cells.
18. Wash each column twice with 1 mL of separation buffer, allowing the first mL to flow through before adding the second.
19. Pellet cells by centrifugation at 200 rcf for 5 min (4ºC).
20. Discard supernatant and resuspend pellet in 100 μL of staining buffer.
21. Count Ter119-negative cells (for NC-3000 cell counter use Solution 13).
Cell staining
1. Using staining solution, adjust the volume of Ter119-negative cells in staining solution to achieve 1 x 107 cells per 100 μL.
2. Gently resuspend staining control and unenriched cells (set aside earlier) by vortexing.
3. Aliquot staining control cells into 9 tubes of 100 µL each.
4. Prepare the Lineage cocktail by combining 3 µL of each antibody in Figure 1.
5. Add the relevant conjugated antibodies to each tube according to Figure 2.
6. Cover stained cells and incubate on ice for 45 min in the dark.
7. Add 1 mL ice-cold staining solution to wash cells.
8. Pellet cells by centrifugation at 200 rcf for 5 min (4ºC).
9. Discard supernatant and resuspend in 1 mL ice-cold staining solution.
10. Pellet cells by centrifugation at 200 rcf for 5 min (4ºC).
11. Re-suspend full stain in 400 μL ice-cold staining solution (+ 5 mM EDTA).
12. Re-suspend controls in 200 μL ice-cold staining buffer (+ 5 mM EDTA).
13. Make a 1:150 dilution of Hoechst in ice-cold staining buffer.
14. Add 1:100 of diluted Hoechst to the relevant tubes immediately prior to sorting (Figure 2).
Cell sorting.
1. Establish voltages and flow using unstained and single stain samples:
A. Gate cells for forward and side scatter to isolate single cells.
2. Establish population gating using flourescence minus one (FMO) samples:
A. Gate out Hoechst-positive cells to remove dead and dying cells.
B. Gate out FITC-positive cells to remove non-erythroid cells.
C. Gate for specific population on levels of CD71 and Ter119 (Figure 3).
3. Sort mature populations into 500 µL collection buffer using unenriched sample.
4. Sort progenitor populations into 500 µL collection buffer using progenitor full stain sample.
1. Socolovsky M. et al. Negative autoregulation by FAS mediates robust fetal erythropoiesis. PLoS Biol. 5, e252 doi:10.1371/journal.pbio.0050252 (2007)
2. Oudelaar A.M. et al. Dynamics of the 4D genome during in vivo lineage specification and differentiation. Nat Commun. 11:2722. doi:10.1038/s41467-020-16598-7 (2020)
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
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