Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Animal procedures were performed in accordance with the approved institutional animal care and use committee of the Institut Pasteur (CETEA).

1. In Utero Pulse Labeling in Csf1r ^MeriCreMer Rosa ^LSL-YFP Embryos

1. Prepare the stock solution of 4-hydroxytamoxifen (OH-TAM, 50 mg/mL).
   1. Under the fumehood, open the 25 mg vial of OH-TAM and add 250 µL ethanol (100%).
   2. Transfer the OH-TAM solution to a 2 mL microcentrifuge tube with a truncated tip and vortex at maximum speed for 10 min.
   3. Sonicate 30 min in a sonicator bath.
   4. Add 250 µL of PEG-35 castor oil under the fumehood to obtain a 50 mg/mL stock solution.
      NOTE: PEG-35 castor oil is a solvent with amphiphilic properties that binds hydrophobic molecules and solubilizes them in aqueous solvents.
   5. Vortex for approximately 5 min at maximum speed and sonicate 30 min in a sonicator bath.
   6. Aliquot 90 µL (4.5 mg) per microcentrifuge tube (1 aliquot per injection).
   7. Store at 4 °C for 1 week or at -20 °C for long term as previously described ¹³.
2. Prepare the stock solution of Progesterone (10 mg/mL)
   1. Add 250 µL of 100% Ethanol to 25 mg of progesterone to prepare a 10 mg/100 µL suspension under the fumehood. Vortex gently.
   2. Add 2250 µL autoclaved sunflower oil to make 10 mg/ml progesterone solution under the hood.
   3. Vortex for approximately 5 min at maximum speed, aliquot and store at 4 °C. Note that the solution is clear when stored.
3. Tamoxifen administration
   NOTE: Embryonic development was estimated considering the day of vaginal plug formation as Embryonic day (E) 0.5. Recombination is induced by single injection at E8.5 into pregnant Csf1r^MeriCreMerfemales ¹². Supplement OH-TAM with 37.5 µg per g of Progesterone to reduce abortion rates after tamoxifen administration. Inject at 1 pm (for vaginal plug observed in the morning).
   1. On the morning of injection, sonicate an aliquot of the OH-TAM solution for 10 min (or until completely resuspended).
   2. Add 360 µL NaCl 0.9% under the fumehood to obtain a 10 mg/mL OH-TAM solution and vortex thoroughly. Sonicate until the solution is clear and completely resuspended (at least 30 min).
   3. Pre-warm progesterone to room temperature.
   4. Mix 450 µL of OH-TAM and 225 µL of progesterone in a microcentrifuge tube. Vortex.
   5. Sonicate at least 10 min and load on to a 1 mL syringe with a 25G needle.
   6. In the animal facility, weigh the pregnant female (Csf1r^MeriCreMer females are in an inbred FVB genetic background and typical weigh between 25 and 33 g).
   7. Perform an intra-peritoneal injection by slowly injecting the calculated volume into the mouse. After withdrawing the needle, gently press on the puncture wound and massage the abdomen to distribute the OH-TAM.
      NOTE: The OH-TAM injection dose is 75 mg/kg and the Progesterone dose is 37.5 mg/kg. Table 1 provides the volume to inject into pregnant females.

Table 1: Injection volume of 4-OH-tamoxifen (OH-TAM). Volume required for a single injection at E8.5 of 75 µg per g (body weight) of OH-TAM supplemented with 37.5 µg per g of progesterone.

2. Dissection of the Yolk Sac (YS) and Fetal Liver (FL)

NOTE: Rigorous sterile techniques are not necessary when manipulating embryos unless if they are going to be used for long-term culture. Nevertheless, the working area must be clean and covered in foil under absorbent paper.

1. Prepare ice-cold phosphate buffered saline (PBS) and digestion mix (PBS containing 1 mg/mL collagenase D, 100 U/mL Deoxyribonuclease I (DNase I) and 3% fetal bovine serum).
2. Sacrifice pregnant females by cervical dislocation at the required gestation day (e.g. embryonic stage E10.5).
3. Pinch the skin just over the genitals and make a small incision at the midline with scissors. Pull the skin toward the head to expose completely the body wall without fur. Cut the abdominal muscles to expose the internal organs and push the gut to expose the two uterine horns.
4. With middle-sized blunt forceps, grab the fat-pad attached to the ovary and gently pull the uterus. Cut at the cervical level of the uterine horns and lift the horns from the peritoneal cavity. Remove the fat-pad to completely free the uterine horns and cut the horns from the ovary on each side.
5. Put the horns into ice-cold PBS in a 10 mm Petri dish. Rapidly grip the uterus muscle layers at one extremity (cervical end) and slide fine scissors between the muscle layer and the decidual tissue to release the embryos with the surrounding decidual tissue.
   NOTE: Muscle tend to rapidly contract after the extraction, so this step must be as quick as possible.
6. Use one pair of fine forceps to cut off the Reichert's membrane and the placenta.
7. Gently remove the yolk sac and place it in a 24-well tissue culture plate with 0.5 mL of digestion mix.
   NOTE: At this step, embryonic blood can be collected.
   1. Immediately after severing the umbilical and vitelline vessels, transfer the embryo into a 12-well tissue culture plate containing 10 mM ice-cold ethylenediaminetetraacetic acid (EDTA). Decapitate the embryo using sharp fine scissors, trying to limit as much as possible tissue dilaceration. Incubate on ice for 10-15 min and collect the EDTA containing the blood.
8. Remove the amnion surrounding the embryo.
9. For stages <E11.5, count the number of somite pairs for better staging of embryos and a better time resolution (each somite pair develops in ~1 h 30 min).
10. Cut the head of the embryo using forceps or fine scissors. Transfer the head to a 24-well plate with 0.5 mL digestion mix.
    NOTE: The neuroectoderm and brain at later stages are further dissected for flow cytometry analysis; carefully remove the surrounding vascular plexus.
11. Cut the embryo above the hindlimb and remove the forelimbs.
12. To isolate the liver, open the thorax using a pair of fine forceps. Pinch anteriorly to the heart and gently pull while using the second forceps to free the organs from the body.
    1. Carefully separate the fetal liver from the heart and gut. Transfer the fetal liver to a 24-well plate with 0.5 mL digestion mix. Carefully monitor the transfer under the dissecting microscope.
13. Collect the tail region (or any other embryo part) for genotyping by polymerase chain reaction assay (PCR).
    NOTE: Other tissues and organs can be harvested from E10.5 embryos for a similar analysis using flow cytometry. Blood, head skin, aorta-gonad-mesonephros region (AGM), heart and neuroectoderm can be collected at E10.5. At later stages, lung, kidney, spleen and pancreas can also be collected. A detailed description of the dissection of the AGM at different developmental stages has been previously described ¹⁴.

3. Processing of Embryonic Tissues for Flow Cytometry

1. Incubate the organs (placed in the digestion mix) for 30 min at 37 °C.
2. Transfer the tissue and enzymatic solution onto a 100 μm strainer placed in a 6-well tissue culture plate filled with 6 ml of FACS buffer (0.5% Bovine Serum Albumin (BSA) and 2 mM EDTA in 1x PBS). Mechanically dissociate by gently mashing with the black rubber piston of a 2 mL syringe to obtain a single-cell suspension.
   NOTE: From now on, all steps should be performed at 4 °C.
3. Collect the cell suspension with a Pasteur pipette and transfer into a 15 mL tube.
4. Spin for 7 min at 320 x g at 4 °C. Discard the supernatant by aspiration.
5. Resuspend the pellet in 60 µL Fc-blocking buffer (CD16/CD32 blocking antibody diluted 1/50 in FACS buffer).
   1. Transfer 50 µL of the single-cell suspension per well in a round bottom 96 multi-well plate. Incubate for at least 15 min on ice.
      NOTE: The staining can also be performed in 5 mL polystyrene FACS tubes when handling a small number of samples.
   2. Transfer the remaining 10 µL into a 5 mL polystyrene FACS tube to obtain a pool of the samples from each tissue. This pool will serve as the controls (i.e. unstained samples and fluorescent minus one (FMO) controls for each fluorochrome). Transfer 50 µL of the pool for each fluorescent minus one (FMO) control (one per fluorochrome) to the 96 multi-well plate.
      NOTE: Each FMO control contains all the fluorochrome-coupled antibodies from the antibody panel, except for the one that is being measured. FMOs are used to identify gate boundaries and to control for the spectral overlap in multicolor panels. To correctly address the variations in background and autofluorescence between different tissues, it is important to prepare these controls from the pool of samples of each tissue. The appropriate control for YFP expression will be the samples from Cre-negative embryos, which are confirmed by PCR genotyping.

4. Surface Antigen Staining

1. Prepare the antibody mix in FACS buffer (Table 2). Prepare 50 µL of antibody mix per sample.
   1. Use the following fluorochrome-coupled antibodies: anti-CD45.2 (clone 104); anti-CD11b (clone M1/70); anti-F4/80 (clone BM8); anti-AA4.1 (clone AA4.1); anti-Kit (clone 2B8); and anti-Ter119 (clone Ter119).
   2. Prepare six antibody mixes for the FMO controls in FACS buffer. Prepare 50 µL of antibody mix per control sample.
      NOTE: The antibody dilution in the antibody mix is two times more concentrated than the final concentration indicated in the materials table. For a panel with six fluorochrome-coupled antibodies, 6 FMO controls are prepared. Table 2 provides the composition of the antibody mix and FMO controls for one tube.

Table 2: Volume of antibodies using for staining and Fluorescent minus one (FMO) controls. Volume (µL) of antibody required for a final volume of 50 µL.

2. Add 50 µL of the antibody mix to the samples in the 96-multiwell plate (final volume during staining is 100 µL). Gently pipette up and down twice and incubate on ice for 30 min.
3. Spin the 96-multiwell plate for 7 min at 320 x g at 4 °C and discard the supernatant. Resuspend in 200 µL of FACS buffer.
4. Repeat wash procedure (step 4.3) twice with 150 µL FACS buffer.
5. Filter the samples and the controls (FMOs and unstained pool samples) into 5 ml polystyrene tubes through a 70 μm strainer. Store on ice until acquisition.

5. Identification of EMPs and YS-derived Macrophages by Flow Cytometry

NOTE: This protocol was optimized using a 4 laser flow cytometer equipped with a 405 nm Violet laser, a 488 nm Blue laser, a 562 nm Yellow laser and a 638 nm Red laser.

1. Prepare compensation beads for each coupled antibody following the manufacturer's instructions. Use unstained samples and compensation beads to optimize laser intensities.
2. To identify gate boundaries, use FMO (Fluorescence minus one) controls for each antibody.
3. In order to exclude dead cells, add 1 µL of DAPI (1 mg/ml) to the tube (containing 200 µL of stained cell suspension) 1 min before acquisition. Use the strong DAPI signal and the forward-scattered parameter (FSC) to perform exclusion of dead cells and debris.
   NOTE: Use software from the flow cytometer to draw gates during acquisition. Compensation matrix and analysis can be performed after sample acquisition using other commercially available software.
4. Use forward and side scatter (FSC vs SSC) to perform live cell and doublet discrimination from total events.
5. Create fluorescence dot plots in log-scale axis and draw daughter gates to, first, exclude erythrocytes (Ter119⁺) and, then, to identify progenitor cells (Kit⁺) and hematopoietic cells (CD45⁺ Kit^neg) (See Figure 1).
6. Create fluorescence histograms to quantify the labeling efficiency of YFP among the different identified populations in the YS (Figure 2), fetal liver (Figure 3) and brain (Figure 4).