This protocol allows for the identification and isolation of BFU-E and CFU-E erythroid progenitors directly from freshly-harvested mouse tissue like bone marrow and spleen. Using this technique, we can isolate pure populations of erythroid progenitors, which was not possible with the previous flow protocols. This method greatly enhances our ability to study erythroid disease mouse models by allowing progenitor isolation for many downstream experiments.
To begin, place the freshly dissected femurs and tibia directly into cold staining buffer or SB-5 and keep the tubes on ice until ready to extract the bone marrow. Place a clean sterilized mortar on ice in a bucket and transfer the bones to the mortar. Using the dissection scissors, snip off approximately 1-millimeter ends of each bone.
Use a 3-milliliter syringe equipped with a 26 gauge needle containing SB-5 to flush the marrow out of the bones and collect it into the mortar and repeat the process 3 to 5 times using fresh buffer each time until the bones appear colorless. Filter the flushed marrow through a 100-micrometer mesh and collect the cells in a 50-milliliter centrifuge tube placed on ice. Next, use the mortar and pestle to crush the flushed bones in 5 to 10 milliliters of SB-5.
Filter the mixture of crushed bones and buffer through the 100-micrometer cell strainer to pool with the previously collected cells. Set up a 100-micrometer mesh filter on an empty 50-milliliter centrifuge tube placed on ice. Place a single spleen on the mesh filter and add 0.5 to 1 milliliter of SB-5 to the spleen to ensure it does not dry.
Using the rubber side of a 3 or 5-milliliter syringe plunger, mash the spleen through the mesh. Add more SB-5, 5 milliliters at a time, and continue to mash until all the cells have been collected in the tube. Prepare a single-cell suspension by pipetting up and down the collected cells with 2 milliliters of SB-5 using a large orifice tip.
Stain the fluorescence minus one or FMO controls by adding all the antibodies except the FMO's namesake antibody to the cell suspension in the FACS tube. To stain the single-color controls, add a single antibody to the cell suspension in the FACS tube. Vortex each control tube for 2 to 3 seconds at 3000 RPM and then incubate at 4 degrees Celsius rocking for 2.5 hours in the dark.
After the incubation, wash the cells with SB-5 and make up the volume of the cell suspension to 4 milliliters with SB-5. Spin down the cells at 900 g for 10 minutes at 4 degrees Celsius and aspirate the supernatant. Re-suspend the unstained and all the single-color controls in 300 microliters of SB-5 and filter through a 40-micrometer filter mesh into new FACS tubes.
Make a DAPI SB-5 solution by diluting the DAPI stock at a ratio of 1 to 10, 000 in SB-5. Re-suspend the FMOS in 1.8 milliliters of DAPI SB-5 and DAPI single-color control in 300 microliters of DAPI SB-5, then filter them through a 40-micrometer filter mesh into new FACS tubes. After adding the antibody master mix to each sample, vortex the tubes for 2 to 3 seconds at 3000 rpm followed by incubation at 4 degrees Celsius in the dark for 2.5 hours in the rocking condition.
After washing the cells as demonstrated previously, re-suspend the samples in 1.8 milliliters of DAPI SB-5 and filter through a 40-micrometer filter mesh into a new FACS tube and analyze the samples using a cystometer. Use the forward scatter to remove the debris based on size, then use the forward scatter height versus forward scatter area histogram to exclude cell aggregates and select single cells. Repeat the exclusion with the side scatter height versus side scatter area histogram.
Exclude the dead cells by gating out the positive cells for DAPI. Select the lineage negative TUR one 19 negative kit positive cells. And from these select a subpopulation that expresses CD 55.
Subdivide the lineage negative TUR one 19, negative kit, positive, CD 55 positive cells into 2 principle populations, P six and P seven based on the expression of CD 49 F and CD 1 0 5. Now based on the expression of CD 150 and CD 41, subdivide P 6 further into P 3, P 4 and P 5. Similarly, based on the expression of CD 150 and CD 71, subdivide P 7 further into BFUE, early CFUE and late CFUE.
In the present study, burst and colony forming units were identified from the freshly harvested bone marrow and spleen cells. After 72 hours of erythropoietin, or vehicle injection in the mice, erythropoietin stimulation showed expansion of the early and late colony forming unit populations P 1 low and P 1 high in the harvested bone marrow and spleen cells. The response of bone marrow and spleen progenitors to erythropoietin in vivo also showed a higher number of cells in the early and late colony forming unit populations P 1 low and P 1 high, compared to vehicle control.
It is most important to generate a single cell suspension before labeling the cells with antibodies. This can be achieved by pipetting up and down repeatedly and by including EDTA in the buffer. The purified progenitors can be used for any downstream cellular and molecular analysis.
For example, single cell transcriptomics, atesque, colony assays and biochemistry. This technique helped us test the predictions from single cell RNA-seq experiments.
