Culturing and Harvesting of HSPC Clones: A Method to Obtain the Singly-Sorted HSPC Clones

Abstract

Source: Huber, A. R., et al. Characterizing Mutational Load and Clonal Composition of Human Blood. J. Vis. Exp. (2019).

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

1. 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.
2. Prepare 50 μL of 2x HSC staining mix according to the recipe seen in Table 1
3. Mix 50 μL of cell solution with the prepared HSC stainin....

Results

Figure 1: Flowchart depicting experimental procedure based on input material. Please click here to view a larger version of this figure.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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. coli-expressed)	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. Commercially available PBS can also be used
Primocin	Invivogen	ant-pm-1	Antibiotic formulation