This video describes the protocol for isolating HSPCs from the bone marrow and sorting them using a fluorescent activated cell sorting technique, which is a method to obtain singly-sorted HSPCs. We culture these sorted cells to obtain HSPC clones.
Protocol
1. HSPC Isolation, Sorting, and Culture Spin down at 1–2 x 107 mononuclear cells for 5 min at 350 x g and resuspend in 50 μL of FACS buffer. Resuspend the cell pellet in 100 μL of anti-CD3 staining solution (1:100 dilution of anti-CD antibody in FACS buffer). Transfer the cells to a microcentrifuge tube. NOTE: When sorting with >2 x 107 cells, increase the antibody mix and FACS buffer volumes accordingly. …
Representative Results
Figure 1: Flowchart depicting experimental procedure based on input material. Please click here to view a larger version of this figure. Figure 2: Cell sort…
Divulgations
The authors have nothing to disclose.
Materials
CD11c FITC
BioLegend
301603
Clone 3.9
CD16 FITC
BioLegend
302005
Clone 3G8
CD3 BV650
BioLegend
300467
Clone UCHT1
CD34 BV421
BioLegend
343609
561
CD38 PE
BioLegend
303505
Clone HIT2
CD45RA PerCP/Cy5.5
BioLegend
304121
Clone HI100
CD49f PE/Cy7
BioLegend
313621
Clone GoH3
CD90 APC
BioLegend
328113
Clone 5E10
Cell Strainer 5 mL tube
Corning
352235
CELLSTAR plate, 384w, 130 µL, F-bottom, TC, cover
Greiner
781182
Human Flt3-Ligand, premium grade
Miltenyi Biotech
130-096-479
Reconsititute in single-use aliquots (25 μL) at 100 μg/mL in 0.1% BSA in PBS
Human Recombinant IL-3 (E. coli-expressed)
Stem Cell Technologies
78040.1
Reconsititute in single-use aliquots (2.5 μL) at 100 μg/mL in 0.1% BSA in PBS
Human Recombinant IL-6 (E. coliexpressed)
Stem Cell Technologies
78050.1
Reconsititute in single-use aliquots (5 μL) at 100 μg/mL in 0.1% BSA in PBS
Human SCF, premium grade
Miltenyi Biotech
130-096-695
Reconsititute in single-use aliquots (25 μL) at 100 μg/mL in 0.1% BSA in PBS
Human TPO, premium grade
Miltenyi Biotech
130-095-752
Reconsititute in single-use aliquots (12.5 μL) at 100 μg/mL in 0.1% BSA in PBS
Lineage (CD3/14/19/20/56) FITC
BioLegend
348701
Clones: UCHT1, HCD14, HIB19, 2H7, HCD56
PBS
Made in at Institute's facility. Commerically available PBS can also be used