HSC and HPC staining and sorting for RNA, Western blot, transplantation or ATAC_seq
Isolate tibias, femurs and when sorting, pelvis and spine. For splenocytes, macerate between two frosted slides.
Flush tibias and femurs with a 25 gauge needle and 5 mL Luer lock syringe. Suspend cells in 3 mL of staining media (magnesium/calcium free HBSS + 2% bovine serum). Pass through a 100 micron filter.
Count cells on a hemacytometer.
For analysis of HSC/HPC frequency in hindlimbs, remove 800 µL of cells and spin. Resuspend in staining media + antibodies. (CD150, CD48, lineage, c-kit, Sca1 and CD34 in some cases). Try and keep CD150 on the PE channel because this antibody works better than other colors, for unclear reasons. Lineage stains should include CD2, CD3, CD8, Gr1, B220 and Ter119, but exclude CD11b and CD4 as these are expressed at low levels on fetal HSCs. Stain 800 mL cells in 200 µL of staining media.
Wash by filling the FACS tube with staining media and spinning 5 min at 1500 RPM.
Resuspend in DAPI media (1:1000). Analyze on Aria or Fortessa.
Isolation of HSCs/HPCs by flow cytometry
Harvest hindlimbs, pelvis and spine. Crush bones with a mortar and pestle in 5 mL of staining media. Pass through a 40 micron filter into a 50 mL Falcon tube. Repeat twice and wash an additional time with staining media.
Spin Falcon tubes for 5 min at 1500 RPM. Aspirate supernatant and resuspend cells in 800 µL with biotin conjugated c-kit antibody (1:200). Incubate 15 min on ice. Wash by adding 10 mL of staining media.
Spin and resuspend with 800 µL of anti-biotin beads (Miltenyi). Use beads at 1:10 dilution. Wash by adding 10 mL of staining media.
Spin and resuspend in 5 mL of staining media. Transfer to 15 mL Falcon tube and separate cells on the Automacs. (for lower cell numbers, this can be done in lower volumes with 5 mL FACS tubes).
After kit selection, spin selected cells for 5 min at 1500 RPM.
Resuspend pellet in 200 µL of staining media with antibodies (CD150 PE, Lineage FITC, CD48 APC, Percp5.5 Sca and streptavidin PE/CY7 – or equivalent staining cocktail as needed).
Wash with 5 mL of staining media and resuspend in 1 mL staining media with DAPI (1:1000).
Sort cells on Aria. Sort into 0.5 mL of staining media in an eppendorf tube on yield mode for the first sort. Collect as many cells as possible. (except for transplantation sorts, see below).
Transfer sorted cells to a FACS tube and perform second sort on purity mode.
For RNA collection
Aim to collect 20,000 double sorted cells (though less will work) in 0.5 mL of PBS + 0.2% BSA. Spin cells and pipet off all but a few microliters of the supernatant. Resuspend in 350 microliters of RLT-plus buffer (with betamercaptoethanol added at 1:100). These samples can then be stored at -80C. When RNA is made, use the RNAeasy micro PLUS kit from Qiagen for the low sample size and gDNA removal columns.
For Western blot
Perform second sort directly into 50 microliters of TCA or, alternatively, into staining media. Westerns work best with 20K-30K sorted cells, depending on the antibodies. Follow the Western blot protocol.
For ATAC seq/Chipmentation
Sort 50,000 cells into PBS + 0.2% BSA. Spin down cells and proceed to ATAC-seq protocol. Likewise, for Chipmentation sort 10,000-50,000 cells and proceed to Chipmentation protocol.
Perform only single sorts on purity mode. Two passes through the sorter seems to compromise HSC function.
Isolate 20 HSCs into 96 well plate. Each well should have 100 µL of staining media and 300,000 LY5.1 competitor cells preloaded.
After sort, draw up contents of each well into separate insulin syringes. For each syringe, draw up the 100 µL, then wash with 30 µL staining media and draw that into syringe as well. Inject contents of the entire syringe into retroorbital sinus.