Preparing PVA-based HSC media:
1. Media should be prepared fresh for every use and media should be pre-warmed to 37°C before use.
2. Mix media reagents to make F12 media supplemented with 10mM HEPES, 1X P/S/G, 1X ITSX, 1 mg/ml PVA, 100 ng/ml TPO, and 10 ng/ml SCF. Prepare enough for 200 ul per well. Note that PVA solution is viscous and will need to be pipetted slowly. Mix media well by inversion before use.
3. Transfer 200 ul media into desired 96-well plate wells (or 1 ml media for 24-well plate wells). Fill remaining plate wells with PBS.
Isolating c-Kit+ cells from mouse bone marrow:
1. Count mouse bone marrow cells and spin down at 440 g for 5 minutes at 4°C.
2. Resuspend cells in 107 cells/ml in PBS and add anti-c-Kit Microbeads at a ratio of 20 ul per 107 cells.
3. Mix and incubate at 4°C for 15 minutes, then add 10 ml of PBS and spin down at 440 g for 5 minutes at 4°C.
4. Resuspend in 2 ml PBS and perform magnetic column enrichment using LS columns, according to the manufacturers’ protocol.
5. Count the retrieved c-Kit-enriched cells by transferring 10 ul of the cells to a tube, mixing 1:1 with Trypan Blue and count using a hemocytometer.
6. Spin down cells at 440 g for 5 minutes at 4°C and resuspend in at 1 x 106 cells/ml. Seed 1 ml per 48-well plate well.
7. Fill any unused wells with PBS and incubate at 37°C with 5% CO2 and 20% O2.
Maintaining HSPC cultures:
1. Media changes must be performed every 2-3 days throughout the entire culture using pre-warmed and freshly-prepared media. Inspect the cell cultures before performing each media change using a light microscope.
2. Collect all the cell culture media in the well into a 15 ml tube using a pipette.
3. Spin down at 440 g for 5 minutes at RT. After removing supernatants, add new media and resuspend in at ~1 x 106 cells/ml. Seed 1 ml per well in 48-well plate.
4. After media changes, transfer back to the tissue culture incubator.
5. Once wells exhibit 80-90% confluency, cell cultures can be passaged at a ratio of 1:2-1:3.
6. Cells cultures can be analyzed at any time point as described below. Alternatively, cell cultures can be used in in vivo transplantation assays (see other published protocols on the HSC transplantation assay) (4,7,8).
Analyzing HSPC cultures:
1. To count the cell cultures, gently pipette the cultures to dissociate attached cells. Transfer 10 ul of the culture to a tube. Mix 1:1 with Trypan Blue and count using a hemocytometer. Alternatively, an automated cell counter can be used.
2. To analyze by flow cytometry, cells should be first stained with Sca1, cKit, and Lineage antibodies: Dissociate attached cells by gently pipetting the media over the plate bottom. Transfer cells to a tube and spin down at 1500 rpm (440 g) for 5 minutes.
3. Resuspend cells in PBS containing the antibodies against CD150, c-Kit, Sca-1, CD4, CD8, CD45R/B220, Ter119, Ly6G/Ly6C, CD127, and CD11b. Stain cells at 4°C for 30 minutes, wash with at least 10 volumes of PBS and spin down.
4. Resuspend cells in ~200 ul PBS containing 1X PI, transfer to a FACS tube, and store at 4°C until analysis.
5. Analyze on a FACS machine/flow cytometer using unstained and single stained samples as controls, and quantify the frequency of CD150+Kit+Sca1+Lineage- live cell population.