CD34 + cells were isolated from mononuclear cells derived from mobilized peripheral blood stem cells (PBSC) autologous transplant products by using EasySep Human CD34 Positive Selection Kit II (StemCell Technologies, 18056). Cell were cultured for 48 h before electroporation in StemSpan™ Serum-Free Expansion Medium II (SFEM II) (StemCell Technologies, 09605) with streptomycin (20 mg/mL), penicillin (20 unit/mL) and the following human cytokines (all from GenScript unless stated otherwise, catalog numbers and dilution used in parentheses): FLT3L (Z02926, 100 ng/mL), G-CSF (Z02980, 10 ng/mL), SCF (Z02692, 100 ng/mL), TPO (Pepro-Tech, 300-18, 25 ng/mL) and IL-6 (Z03034, 10 ng/mL). Cells were cultured at a density of 2.5*10^5 cells/ml in 96-well U-bottom plates.
Stemspan serum free expansion medium 2 sfem 2
Manufactured by STEMCELL
Sourced in United Kingdom
StemSpan™ Serum-Free Expansion Medium II (SFEM II) is a cell culture medium designed for the expansion of hematopoietic stem and progenitor cells in a serum-free environment. It provides the necessary nutrients and growth factors to support the proliferation and maintenance of these cell types.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by STEMCELL (1 mentions)
G csf, by Thermo Fisher Scientific (1 mentions)
Flt3l, by Thermo Fisher Scientific (1 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (1 mentions)
Easysep human cd34 positive selection kit 2, by STEMCELL (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Anti pe microbeads, by Miltenyi Biotec (1 mentions)
Napabucasin, by MedChemExpress (1 mentions)
Nutlin 3a, by Merck Group (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Steady glo luciferase assay system, by Promega (1 mentions)
Cytotune 2, by Thermo Fisher Scientific (1 mentions)
4 protocols using stemspan serum free expansion medium 2 sfem 2
1
Expansion and Electroporation of CD34+ Cells
2
Megakaryocytic Precursor Isolation and Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Megakaryocytic precursors were purified using Magnetic Activated Cell Sorting (MACS) kit with anti‐PE microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany). Briefly, cells were incubated with anti‐CD41a‐PE antibody diluted 1:20 for 15 minutes. Cells were washed, re‐suspended in CATCH buffer and incubated with anti‐PE microbeads. Unbound microbeads were removed by washing in CATCH buffer. Re‐suspended cells were filtered through a pre‐wet 100 μm nylon mesh and passed through equilibrated LS MiniMACS columns. Labelled cells were eluted, pelleted by centrifugation, re‐suspended in PBS and cultured in StemSpan Serum‐Free Expansion Medium II (SFEM II; StemCell Technologies, Vancouver, BC) containing 30 nmol L−1 TPO (Thermo‐Fisher Scientific) for 3 days.
3
Co-culture of B-ALL PDX with MSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For co-culture experiments with Luciferase-expressing B-ALL PDX samples, 3x104/cm2 mesenchymal stem cells (MSC) were plated in 96-well tissue culture plates and 2x104 B-ALL PDX cells were added in StemSpan Serum-Free Expansion Medium II (SFEM II, STEMCELL Technologies, Cambridge, UK).17 (link),18 (link) Cells were treated for 5 days with indicated drugs and luminescence analyzed with the Steady-Glo Luciferase Assay System (Promega, Southampton, UK), according to manufacturer’s instruction, and detected with Infinite m200 Pro microplate reader (Tecan, Reading, UK). The following reagents and inhibitors were used, S3I-201 (Cayman Chemical, MI, USA), Napabucasin (BBI608; MedChemExpress, NJ, USA), C188-9 (MedChemExpress and Adooq Biosciences, CA, USA), Nutlin-3a (Merck Life Science, Dorset, UK) and Idasanutlin (Bio-Techne, Abingdon, UK).
4
Erythroblast Reprogramming and iPSC Generation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CRO1 refers to a young female individual with DS caused by a microduplication of a 4.083 Mb region overlapping the DSCR. Patient blood sample was collected, as described above, during routine diagnostic tests, and consented surplus material was used to generate the iPSCs.
Extracted PBMCs were cultured in Erythroblast differentiation medium (StemSpan Serum-Free Expansion Medium II (SFEM II; StemCell Technologies) supplemented with: 2 U/mL Human Erythropoietin, 50 μg/mLl -ascorbic acid, 50 ng/mL Stem Cell Factor, 40 ng/mL IGF-1, 10 ng/mL IL-3, 0.4 ng/mL Dexamethasone, 1% l -glutamine and 1% MEM Non-Essential Amino Acids) for 10 days to allow growth and expansion of erythroblasts. Erythroblasts were reprogrammed using the iPS 2.0 Sendai Reprogramming Kit comprising three reprogramming vectors containing the four Yamanaka factors: KOS (human Klf4 and human Oct3/4, Sox2), hc-Myc (human c-Myc) and hKlf4 (human Klf4) (CytoTune2.0, Invitrogen, Thermo Fisher). Individual colonies were expanded and two clones were selected for experimental use after pluripotency and genome validation.
Extracted PBMCs were cultured in Erythroblast differentiation medium (StemSpan Serum-Free Expansion Medium II (SFEM II; StemCell Technologies) supplemented with: 2 U/mL Human Erythropoietin, 50 μg/mL
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!