The overall goal of this experiment is to characterize yolk sac hematopoietic progenitors and their progeny in vivo using a mouse tamoxifen induced fate mapping system and flow cytometry. This can help answer key questions in the immunology field concerning the development and differentiation of hematopoietic progenitors that are distinct from hematopoietic stem cells. The main advantage of this technique is that the hematopoietic progenitors labeled within a limited time window and the progeny such as resident macrophages can be monitored during the development and adulthood.
Generally, individuals new to this method will struggle because of the deal of cells for embryonic tissue and the large volume of the simultaneously treated samples. To begin the experiment, first transfer dissected mouse uterine horns into a 10 millimeter petri dish filled with approximately 30 milliliters of ice cold PBS. Immediately after, grasp the uterus muscle layers at the cervical end of the horn and slide fine scissors between the muscle layer and a decidual tissue to release the embryos.
Then with fine forceps remove Reichert's membrane and remove the placenta. Gently dissect the yolk sac and transfer it to a 24-weld tissue culture plate filled with 0.5 milliliters of the digestion solution. After severing the umbilical and vituline vessels immediately transfer the embryo into a 12-weld tissue culture plate filled with 2 milliliters of 10 millimolar ice cold EDTA.
After removing the head from the embryo with sharp fine scissors, incubate the body and the head on ice for 10 to 15 minutes and then remove the embryo and collect the EDTA containing the blood. Next, remove the amnion surrounding the embryo. Afterward remove the hind limbs and foreign limbs from the embryo.
Using a pair of fine forceps, open the thorax. Then, grasp the anterior of the heart and gently pull the tissue while releasing the internal organs from the body with a second pair of forceps. Under a dissecting microscope, separate the fetal liver from the heart and gut.
And transfer the organ to a 24 well plate filled with 0.5 milliliters of the digestion solution. Anatomical connection with the cardiovascular system is critical for fetal liver identification. The fetal liver is still in this developmental stage and the younger the embryos are, the more difficult the dissection can be.
To prepare primary cell suspensions, incubate the collected organs in the digestion solution at 37 degrees Celsius for 30 minutes. Load the tissue in the enzymatic solution into a 10 micrometer strainer installed over one weld of a six-weld tissue culture plate filled with 1 milliliter of FACS buffer. Use a black rubber piston from a 2 millimeter sysringe to dissociate the tissue by gently mashing to obtain a single cell suspension.
Then, aspirate the cell suspension with a Pasteur pipette and transfer it into a 15 milliliter tube. Centrifuge the suspension at 320 times G at 4 degrees Celsius for 7 minutes. After centrifugation, aspirate and discard the supernatant.
Next, prepare fresh Fc blocking buffer and the resuspend the pellet in 60 microliters. Then, transfer 50 microliters of the single cell suspension into a well of a 96-well round bottom plate. Incubate the plate on ice for at least 15 minutes.
Next, transfer the remaining 10 microliters of the suspension into a 5 milliliter polystyrene FACS tube to obtain a pool of samples derived from different tissues. Transfer 50 microliters of the pool for FMO controls for each fluorochrome to the 96 multi well plate. To proceed with antigen surface staining, use a set of six flourochrome antibodies to prepare an antibody solution and FACS buffer.
Then prepare six antibody solutions and FACS buffer used for the FMO control staining. Afterward add 50 microliters of the antibody solutions to the samples in the 96 multi well plate. Mix the samples by gentle double pipetting and then incubate the plate on ice for 30 minutes.
Centrifuge the 96 multi well plate at 320 times G at 4 degrees Celsius for 7 minutes and remove the supernatant. Wash the cells in 200 microliters of FACS buffer and spin the plate one more time. Special care should be taken when removing the supernatant by plate inversion.
The 96 multi-well plate must be inverted with a single sharp wrist motion to avoid loosening the cells. Finally, using a 70 micro strainer, filter the tissue and control samples into 6 millimeter polystyrene tubes. Flow cytometric analysis can now be carried out.
A population of a live non-erythrocytic cytic single cells was analyzed for the expression of the progenitor marker kit and the hematopoietic cell marker CD45. Within the targeted population, three different cell sub populations were distinguished. KIT positive CD45 negative, KIT positive CD45 low and KIT negative CD45 positive.
Among the yolk sac kit positive progenitors, both CD45 negative and CD45 low cell populations contained AA4.1 positive cells that express the yellow fluorescent protein or YFP, indicating that immature KIT positive progenitors first express AA4.1 irrespective of CD45 expression. AA4.1 positive, YFP positive cells in the liver and the brain were only found in the KIT positive CD45 low progenitor population and correspond most likely to circulating progenitors that originated from the yolk sac progenitors. Macrophages were found in the kit negative CD45 positive population and were identified as F480 bright CD11 B positive cells.
Macrophages in the yolk sac, liver and brain were efficiently labeled by a single 4-hydroxy tamoxifen administration that may indicate their ENP origin. Once mastered, the procedure for cell isolation and staining can be done in three to four hours if it's performed properly. While attempting this procedure, it's important to remember to always keep samples on ice as soon as they are collected.
Fresh FACS buffers should be prepared daily and filtered. Following this procedure, other mash reporter or flux strains like Rosa MTMG or Rosa confetti lines can be used in order to answer additional questions on the indifferentiation and proto phase potential of the post labeled cells.
