Isolation of Healthy F4/80+ Macrophages from Embryonic day E13.5 Mouse Fetal Liver Using Magnetic Nanoparticles for Single Cell Sequencing

Materials and Reagents

1. 70 μm cell strainer (Fisher Scientific, Falcon, catalog number: 10788201)

2. Round bottom polystyrene 5 ml test tubes (Fisher Scientific, Falcon, catalog number: 14-959-5)

3. 1.7 ml microfuge tubes (Crystalgen)

4. 50 ml conical tubes (BD)

5. Sterile pipettes (Corning), tissue culture 6 cm dishes (BD)

6. Mouse E13.5 embryonic fetal liver

7. EasySep mouse PE positive selection kit (Cell Signaling Technologies, catalog number: 17656)

8. Anti-F4/80-PE antibody (rabbit polyclonal) (eBiosciences, catalog number: 12-4801-80)

9. FWV ICAM4/αv inhibitor peptide (custom sequence LRQGKTLRG) (Genscript, catalog number: SC1848). A stock solution was prepared at 23 mM and stored at -20°C. Stock solution is stable for at least 2 years.

10. Cell Culture Phosphate Buffered Saline 1× (Fisher Scientific, Corning, catalog number: MT21040)

11. Fetal Bovine Serum (GE Healthcare, HyClone, catalog number: SH30070.03)

12. 0.5 M EDTA stock solution

Equipment

1. Pipettes (P200)

2. Dissection tools (e.g., sharp tweezers)

3. EasyEights EasySep Magnet (Cell Signaling Technologies, catalog number: 18103)

4. Refrigerated centrifuges capable of fitting 1.7 ml and 50 ml tubes

5. Flow Cytometer (Attune)

Procedure

1. Mouse E13.5 embryonic fetal liver extraction and cell homogenization

   1. Sacrifice the mouse and extract E13.5 embryos, placing them in ice-cold 1× PBS in a sterile 6 cm tissue culture dish.

   2. Using sharp tweezers, carefully extract the fetal livers into 200 μl of 1× PBS with 2% FBS.

   3. Using a P200, pipette repeatedly but gently to homogenize the livers and generate single cell suspensions (collagenase or other enzymatic treatments are not required).

   4. Filter the cells by passing them through a 70 μm mesh filter into 5 ml of ice-cold 1× PBS + 2% FBS in a 50 ml conical tube.

   5. Mix well, take a small aliquot for counting the cells, and centrifuge the remaining cells in a refrigerated centrifuge at 180 × g for 5 min at 4°C.

      
      

2. F4/80-PE+ cell selection using EZ-Sep mouse PE selection kit

   1. Resuspend the cells in 1× PBS + 2% FBS + 1 mM EDTA (EZ-Sep recommended medium) at room temperature and at a density of up to 10⁸ cells per ml in a microfuge tube. (Usually, each fetal liver generates about 5-10 million cells, so each liver can be considered one sample). We usually resuspend each fetal liver in 200 µl of 1× PBS + 2% FBS in separate tubes.

   2. Add FWV peptide to a final concentration of 2 mM and mix well (~17.5 µl of 23 mM stock solution added per 200 µl of sample).

      Note: At this point onwards, the steps refer to the protocol for EZ-Sep mouse PE selection kit.

   3. Add 2 µl of mouse FcR blocker from the EZ-Sep kit and pipette to mix cells.

   4. At a dilution of 1:100 (based on the total volume from Step B1), add anti-F4/80-PE antibody, mix well by pipetting gently, and incubate at room temperature in a dark place for 20 min.

      Note: Antibody dilutions will vary depending on the antibody used and must be standardized for different antibodies.

   5. Add 2 µl of PE selection cocktail from the EZ-Sep kit, mix well by pipetting gently, and incubate at room temperature for 20 min.

   6. Add 10 µl of magnetic nanoparticles from the EZ-Sep kit, mix well by pipetting gently, and incubate at room temperature for 10 min

   7. Place the tubes on an EasyEights EasySep Magnet from Cell Signaling Technologies (which allows for eight tubes to be processed at once) for 5 min at room temperature.

   8. Gently decant the supernatant by inverting the magnet with the tubes into a waste container (use one smooth inversion motion, do not shake).

   9. Remove the tubes from the magnet and collect the cells in 1 ml of cold EZ-Seq recommended medium by gentle pipetting along the walls of the tubes. Centrifuge at 180 × g for 5 min at 4°C.

   10. Resuspend in 200 µl of EZ-Sep recommended medium at room temperature, and repeat the magnetic nanoparticle selection steps from Step B6.

   11. Remove the tubes from the magnet and wash twice in ice-cold 1× PBS. For each wash, add 2 ml of 1× PBS along the walls of the tubes and place on the magnet for 5min. Decant the liquid gently each time, as in Step B8.

   12. After the final wash, resuspend the cells in 0.5 ml 1× PBS and proceed with single cell sequencing.

   13. Take out a small aliquot of cells and analyze using flow cytometry for the purity of F4/80-PE+ cells.

Data analysis

Please refer to Mukherjee et al. (2021) for details of flow cytometry data analysis of purified F4/80+ population. In this study, 28,000 healthy F4/80+ macrophages (visually counted by light microscopy after Trypan Blue staining) were isolated from two independent wild-type E13.5 fetal livers and pooled for single cell sequencing. For details of single cell sequencing quality, cell heterogeneity, and data analysis, please refer to Mukherjee et al. (2021) . We obtained 13 independent clusters after U-MAP analysis using the Seurat package (Figure 2).

Acknowledgments

The authors would like to thank Xue Li for her input in designing an early version of this protocol. Flow cytometry was performed at the Sinai Flow Cytometry Core. This work was supported by NIH grants R01 DK102260 and DK121671 to JJB and by a Black Family Stem Cell Institute (ISMMS) Pilot Grant to KM.

This protocol was adapted with minor modification from previous study published by Mukherjee et al. (2021; Doi: 10.7554/eLife.61070).

Competing interests

The authors declare no competing interests

Ethics

This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#18-1911) of the Mount Sinai School of Medicine.

References

1. Klei, T. R., Meinderts, S. M., van den Berg, T. K. and van Bruggen, R. (2017). From the Cradle to the Grave: The Role of Macrophages in Erythropoiesis and Erythrophagocytosis. Front Immunol 8: 73.
2. Li, W., Wang, Y., Zhao, H., Zhang, H., Xu, Y., Wang, S., Guo, X., Huang, Y., Zhang, S., Han, Y., et al. (2019). Identification and transcriptome analysis of erythroblastic island macrophages. Blood 134(5): 480-491.
3. Li, W., Guo, R., Song, Y. and Jiang, Z. (2021). Erythroblastic Island Macrophages Shape Normal Erythropoiesis and Drive Associated Disorders in Erythroid Hematopoietic Diseases. Front Cell and Dev Biol 8: 613885.
4. Manwani, D. and Bieker, J. J. (2008). The erythroblastic island. Curr Top Dev Biol 82: 23-53.
5. Mukherjee, K., Xue, L., Planutis, A., Gnanapragasam, M. N., Chess, A. and Bieker, J. J. (2021). EKLF/KLF1 expression defines a unique macrophage subset during mouse erythropoiesis. Elife 10: e61070.
6. Seu, K. G., Papoin, J., Fessler, R., Hom, J., Huang, G., Mohandas, N., Blanc, L. and Kalfa, T. A. (2017). Unraveling Macrophage Heterogeneity in Erythroblastic Islands. Front Immunol 8: 1140.
7. Xue, L., Galdass, M., Gnanapragasam, M. N., Manwani, D. and Bieker, J. J. (2014). Extrinsic and intrinsic control by EKLF (KLF1) within a specialized erythroid niche. Development 141(11): 2245-2254.