Culturing and Harvesting of HSPC Clones: A Method to Obtain the Singly-Sorted HSPC Clones
Published: April 30, 2023
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 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 | ||
| Primocin | Invivogen | ant-pm-1 | Antibiotic formulation |
Citer Cet Article
Culturing and Harvesting of HSPC Clones: A Method to Obtain the Singly-Sorted HSPC Clones. J. Vis. Exp. (Pending Publication), e20274, doi: (2023).