1×106 CD34+ cord blood stem cells were thawed in a 37°C water bath and pipetted into 20 mL pre-warmed StemSpan SFEM medium (STEMCELL Technologies, #09650). Cells were centrifuged and resuspended into StemSpan medium containing 10% fetal bovine serum (FBS) (Sigma, #F4135). Cells were adjusted to 2.5×104 cells/mL, supplemented with 20 ng/mL huIL-3 (PeproTech, #200–03), 100 ng/mL huSCF (PeproTech, #300–07), 100 ng/mL huFLT3L (PeproTech, #300–19), 50 ng/mL huTPO (PeproTech, #300–18), plated at 0.5×104 cells per well in a 96-well U bottom plate and cultured at 37°C in a CO2 incubator. After 7 days of expansion, cells were harvested and frozen in 80% MEM-α (Gibco, #12 561–056), 10% DMSO (Sigma, #D2650), 10% FBS at 2.6×106 cells/mL. Cells were stored in liquid nitrogen.
Huscf
Manufactured by Thermo Fisher Scientific
The HuSCF is a laboratory instrument used for the cultivation and maintenance of human stem cells. It provides a controlled environment to support the growth and differentiation of stem cell cultures. The HuSCF ensures precise control over temperature, humidity, and gas composition to optimize the conditions for stem cell proliferation and maintenance.
Automatically generated - may contain errors
Lab products found in correlation
α mem, by Thermo Fisher Scientific (2 mentions)
Stemspan sfem medium, by STEMCELL (1 mentions)
Huflt3l, by Thermo Fisher Scientific (1 mentions)
Hutpo, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Hu gmcsf, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Mitomycin c, by Merck Group (1 mentions)
2 protocols using huscf
1
Expansion and Cryopreservation of CD34+ Cord Blood Stem Cells
2
Murine Stromal MS-5 Cells for Cord Blood DC Differentiation
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Murine stromal MS-5 cells were obtained from DMCZ (#ACC 441) and cultured in MS-5 medium (MEM-α, Gibco, #15 070–063, 10% FBS, 1% Pen/Strep, Gibco, #15 070–063, 2 mM Sodium Pyruvate, Gibco, #11 360–039). For in vitro cord blood DC differentiation, MS-5 cells were treated with 10 µg/mL Mitomycin C (Sigma, #M4287) for 3 hours at 37°C to stop cell proliferation. After washing, cells were detached by Trypsin-EDTA (Gibco, #25 300–054) and plated at 2.5×105 cells/mL in 100 µL volume per well in a 96-well F bottom plate. The day after, expanded CD34+ cord blood stem cells were added to the pre-treated MS-5 cells to start DC differentiation. 2.6×106 frozen cord blood stem cells were thawed and washed once. Cells were resuspended in 20 mL MS-5 medium supplemented with 5 ng/mL huGM-CSF (PeproTech, #300–03), 5 ng/mL huIL-4 (PeproTech, #200–04), 40 ng/mL huSCF and 200 ng/mL huFLT3L. 100 µL cord blood stem cells at 1.3×105 cells/mL were added to each well to the plated MS-5 cells in the 96-well F bottom plates and cultured at 37°C in a CO2 incubator. Cells were fed on day 6 with 50 µL/well MS-5 medium supplemented with 12.5 ng/mL huGM-CSF, 12.5 ng/mL huIL-4, 100 ng/mL huSCF and 500 ng/mL huFLT3L. In vitro differentiated cord blood DCs were harvested on day 12 or 13 after initiation of the culture.
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