The overall goal of this procedure is to facilitate the identification of enucleation events with an aurn Erythroblast subpopulation. The first step is to create a population enriched in nucleating events. This is accomplished by in vitro erythropoiesis culture, either long-term starting from low density bone marrow cells isolated from the femurs, tibia, and pelvic bones of a non-stressed mouse or short-term culture.
Starting with ple cytes from the spleen of a mouse subjected previously to phlebotomy in order to mount stress erythropoiesis. The cultured cells at the final step of the culture are fixed, permeated, and stained with the fluorescent markers of interest. These cells are run on an imaging flow cytometer and then analyzed to identify the cells undergoing enucleation in the population of nucleating cells signaling and cytoskeletal components that regulate and mediate the process of enucleation can be identified.
The main advantage of this technique over existing methods such as fluorescent microscopy, is that the significantly larger number of nucleating cells can be observed and analyzed in a short amount of time. This method can help to answer key questions in the erythropoiesis field, such as what molecular mechanisms are involved in erythroblast nucleation. To differentiate erythroid cells from low density bone marrow cells begin by adding one milliliter of culture media to a sterile flow cytometry tube.
Then attach a 25 gauge by five eighths inch needle to a tuberculin syringe and gently flush fresh culture media through the harvested femurs and tibia a few times into the flow cytometry tube. After adjusting cell suspension volume to five milliliters with IMDM, carefully layer onto five milliliters of 1.083 grams per milliliter density gradient cell separation medium in a 15 milliliter tube. Transfer everything down to the two milliliter mark into a new 15 milliliter tube and wash the collected buffy coat with fresh culture media lice the red blood cells in three milliliters of red blood cell lysis buffer for five minutes at room temperature after washing cells in culture media and resus suspending the pellet in the same plate, the cells in a six well cell culture plate at a concentration of five times 10th of the fifth cells per well to a final volume of 2.5 milliliters in growth media and incubate the cells at 37 degrees Celsius five days after plating the culture.
Count the number of erythroblasts in each well of the original culture plate and then transfer each well into corresponding individual 15 milliliter conical tubes. After spinning down the cells, aspirate the supernatants and resuspend the pellets in fresh growth media containing only erythropoietin to a concentration of two times 10 of the fifth cells per milliliter. Then aspirate the supernatant from a plate of MS five cells and add the erythroid cells to the wells.
Two days later, replace the supernatant with fresh growth media without cytokines toul erythroblasts from low density PLE cytes. Use the plunger from a five milliliter syringe to macerate the spleen through a 40 micron strainer into a 50 milliliter tube. Wash the cell strainer with growth media and adjust the final volume of cell suspension to five milliliters.
Then after separating the cell layers by density gradient as just demonstrated, lice the red blood cells and wash the cytes in seven milliliters of culture media. Then Resus suspend the pellet in growth media supplemented with cytokines and incubate the cells at one to five times 10 to the six cells in a final volume of one milliliter per well of a 24 well cell culture plate overnight at 37 degrees Celsius and 5%carbon dioxide. The following day.
Transfer cells to wells containing adhered MS five cells as previously described and analyze after six to eight hours, collect the erythroblasts from the culture wells and fix them in 500 microliters of 3.7%formaldehyde in PBS Resus, suspending the pellet with gentle pipetting. After 15 minutes, spin the cells at 2000 GS for 20 seconds in a micro centrifuge, aspirate the supernatant and re suspend the pellet in 500 microliters of PBS. After spinning down the cells in the micro centrifuge, again, aspirate the supernatant again and place the cells on ice for at least 15 minutes.
Put minus 20 degrees Celsius, stored acetone solutions on ice. Then quickly resuspend and spin down the cells in a series of acetone washes as indicated after the fax buffer wash, incubate the cell pellets in 100 microliters of the appropriate antibody cocktail for 30 minutes at room temperature. Then after washing the cells in fax buffer again, re suspend the sustained pellets in 60 microliters of fax buffer containing drac five and run the samples on an imaging flow cytometer collecting at least 10, 000 events per experimental sample.
Then start the analysis by selecting cells based on their brightfield aspect ratio, the ratio of the length of their minor versus their major axis and their brightfield area, which is indicative of their size. Events with a brightfield area value lower than 20 and higher than 200 are mostly debris and cell aggregates respectively and are excluded from the analysis. The then analyze the single cells based on their value for the gradient RMS parameter, which indicates the sharpness of image create a second gate containing the cells with the gradient RMS value greater than the 50th percentile in order to select the images taken well in focus.
Next gate, the cells based on their size as measured by their brightfield area and their positivity for the erythroid marker TER one 19 as measured by the TER one 19 fluorescent stain mean pixel parameter cells very low or very high for TER one 19 are either non erythroid or remaining cell aggregates respectively and are excluded from the analysis in the next step. The cells are selected based on their DR five aspect ratio intensity, the ratio of the minor versus the major axis of their nucleus and the intensity of their d dr five expression. DR five negative cells mostly enucleated cells such as reticulocytes and red blood cells and cells with a low drag five aspect ratio.
Mostly doublets are excluded from the analysis gait. The drag five positive DNA positive cells, which are mostly erythroblasts at this point. These erythroblasts are then analyzed based on their TER one 19 area, which indicates the size of the cell and their TER one 19 mean pixel area or density of TER one 19 expression, which indicates the brightness of their TER one 19 staining.
Ortho chromatic erythroblasts are recognized as small TER one 19 high cells. Finally, a subpopulation of the ortho chromatic erythroblasts is characterized by their low brightfield aspect ratio and their high delta sentu XY TUR one 19. DR five as defined by the distance between the center of the incipient TUR one 19 positive reticulocyte and the center of the DR five positive nucleus along with antibody against her one 19 and the DNA stain DRAAK five.
The cells are typically also stained to identify the localization of f actin during erythroblast and nucleation. Of note, the progression of enucleation can be visualized as cells with a decreasing aspect ratio. That is cells that become increasingly elongated and with an increasing delta OID xy TUR one 19 drag five are observed.
F actin concentrates at the cleavage furrow during in nucleation and then dissipates once the nucleus is extruded. Other proteins and structures of interest can also be stained with the appropriate antibodies or fluorescent markers to allow imaging of their roles during enucleation. For example, polarized microtubule formation can be visualized in wild type ortho chromatic erythroblasts prior to a nucleation, but not in erythroblasts treated with colchicine.
Inhibition of tubulin polymerization by colchicine diminishes the cell polarization as demonstrated by measuring the parameter delta OID XYB FDR five between the center of the cell body seen in the Brightfield channel and the center of the nuclear staining achieved with DR five. Following this procedure, other methods such as fluorescence microscopy can be performed to answer questions concerning the interactions between different cell types involved in erythropoiesis such as those observed in Erythroblast islands. After watching this video, you should have a good understanding of how to visualize enucleation events in an erythroblast enriched population.
