Table of Materials
| Name of Material/Reagent | Company | Catalog Number |
Comments/Description |
|---|---|---|---|
| 0.4 % Trypan blue solution | Thermo Fisher Scientific | 15250-061 | Dilute working stock to 0.2 % in sterile 1 x PBS |
| 0.5 M UltraPure Ethylene diamine tetra acetic acid, pH 8.0 | Gibco | 15575–038 | |
| 10x Hanks Balanced Salt Solution (HBSS) | Invitrogen | 14185052 | Dilute to 1x with sterile distilled water & pH to 7.2 |
| 2.5 % Trypsin, no phenol red | Thermo Fisher Scientific | 15090046 | Dilute working stock to 1x with sterile 1 x PBS |
| 30μm Pre-separation filters | Miltenyi biotech | 130-041-407 | |
| 35 % sterile Bovine serum albumin | Sigma-Aldrich | A7979 | |
| 7-AAD | Biolegend | 420404 | Used as a live/dead stain to eliminate dead cells from FACS |
| Anti-human CD10-FITC antibody (Clone HI10a) | Biolegend | 312207 | Use 1:20 dilution |
| Anti-human CD11b-FITC (activated) antibody (Clone CBRM1/5) | Biolegend | 301403 | Use 1:5 dilution |
| Anti-human CD123-APC antibody (Clone 6H6) | Biolegend | 306012 | Use 1:20 dilution |
| Anti-human CD14-PE antibody (Clone M5E2) | Biolegend | 301806 | Use 1:20 dilution |
| Anti-human CD19-FITC antibody (Clone 4G7) | BD Biosciences | 347543 | Use 1:5 dilution |
| Anti-human CD235a-APC antibody (Clone GA-R2 (HIR2)) | BD Biosciences | 551336 | Use 1:20 dilution |
| Anti-human CD235a-FITC antibody (Clone HIR2) | Biolegend | 306609 | Use 1:50 dilution |
| Anti-human CD3-FITC antibody (Clone UCHT1) | Biolegend | 300405 | Use 1:20 dilution |
| Anti-human CD34-APC-Cy7 antibody (Clone 581) | Biolegend | 343514 | Use 1:20 dilution |
| Anti-human CD38-PE antibody (Clone HIT2) | Biolegend | 303506 | Use 1:20 dilution |
| Anti-human CD41a-PerCP-Cy5.5 antibody (Clone HIP8) | Biolegend | 303720 | Use 1:20 dilution |
| Anti-human CD45Ra-PE-Cy7 antibody (Clone HI100) | Biolegend | 304126 | Use 1:20 dilution |
| Anti-human CD66b-PE-Cy7 antibody (Clone G10F5) | Biolegend | 305116 | Use 1:20 dilution |
| Anti-human CD7-FITC antibody (Clone CD7–6B7) | Biolegend | 343103 | Use 1:20 dilution |
| Dimethyl sulfoxide (DMSO) | Fisher Scientific | BP231–100 | Filter sterilize before use |
| Dulbecco’s Modified Eagle Medium (DMEM) powder with L-Glutamine | Gibco | 12100046 | Reconstitute 1 packet to make 1 L of DMEM media with sodium bicarbonate, 10% FBS and 1x |
| Fetal bovine serum, Australian source, heat inactivated | Omega Scientific | FB-22 Lot #609716 | |
| Human CD34 microbead kit | Miltenyi biotech | 130-046-702 | |
| Human Thrombopoietin (TPO), research grade | Miltenyi biotech | 130-094-011 | Make a stock of 100μ/mL in 1x PBS + 0.1 % BSA. Use 50 ng/mL for both myeloid differentiation |
| L-Glutamine | Omega Scientific | GS-60 | 2 mM concentration in stimulation medium |
| LS Columns | Miltenyi biotech | 130-042-401 | |
| MACS Multi stand | Miltenyi biotech | 130-042-303 | |
| MidiMACS magnetic separator | Miltenyi biotech | 130-042-302 | |
| MNC fractionation media (Ficoll-Paque PLUS) | GE Healthcare | 17-1440-03 | |
| MS-5 cells | Gift from the laboratory of Gay | ||
| Paraformaldehyde | Sigma-Aldrich | P6148 | Heat 800 mL of 1x PBS in a glass beaker on a stir plate in a chemical hood to ~65°C. Add 10 g of paraformaldehyde powder. To completely dissolve the paraformaldehyde, raise the pH |
| Penicillin & Streptomycin | Gibco | 15140-122 | |
| Poly-L lysine | Sigma-Aldrich | P2636 | Make a 10 mg/mL stock in 1x |
| Recombinant human erythropoietin-alpha (rHu EPO-α) | BioBasic | RC21-15 | Make a stock of 2000 units/mL in 1x PBS + 0.1 % BSA. Use 4 units/mL for myeloid |
| Recombinant human fibronectin fragment (RetroNectin) | Takara | T100B | Use 20μg/mL diluted in sterile 1x PBS to coat wells prior to stimulation of CD34+ HSCs. |
| Recombinant human Flt-3 ligand (rHu Flt-3L) | BioBasic | RC214-16 | Make a stock of 100μg/mL in 1x PBS + 0.1 % BSA. Use 5 ng/mL for myeloid differentiation and |
| Recombinant human interleukin-3 (rHu IL-3) | BioBasic | RC212-14 | Make a stoc k of 100μg/mL in 1x PBS + 0.1 % BSA. Use 5 ng/mL for myeloid differentiation and |
| Recombinant human stem cell factor (rHu SCF) | BioBasic | RC213-12 | Make a stoc k of 100μg/mL in 1x PBS + 0.1 % BSA. Use 5 ng/mL for myeloid differentiation and |
| Serum free medium (X-Vivo-15) | Lonza | 04-418Q | |
| Sodium bicarbonate | Fisher Scientific | BP328-500 | |
| Wright-Giemsa stain, modified | Sigma-Aldrich | WG16-500 | Use according to manufacturer's instructions |
| Equipment | |||
| BD LSR II flow cytometer | BD | ||
| Centrifuge | Sorvall Legend RT | ||
| Light microscope | Olympus |