Donor-derived CD34+ cells(kind gift from K. Rajewsky, MDC Berlin, Germany) were propagated in SFEM II(StemCell Technologies, 09605), SCF, FLT3-L, TPO, IL6(all 100ng/ml; easyexperiments.com), UM171(Selleck, 35nM), SR1(Selleck, 0.75μM), 19-deoxy-9-methylene-16,16-dimethyl PGE2(Cayman, 10μM). CD34+ differentiation towards immature MDSC-like cells was induced by switching culture medium to RHB-A-medium supplemented with human SCF(50ng/ml) and human GM-CSF(100ng/ml) for 7-12 days(cl.#1, cl.#2), prior to co-culture.
Um171
Manufactured by Selleck Chemicals
Sourced in Germany
The UM171 is a compact and versatile laboratory equipment designed for various scientific applications. It serves as a general-purpose instrument capable of performing a range of analytical and experimental tasks. The UM171 is known for its reliable performance and ease of use, making it a valuable tool in many research and testing environments.
Automatically generated - may contain errors
Lab products found in correlation
Stemspan sfem 2 medium, by STEMCELL (2 mentions)
Hflt3l, by Thermo Fisher Scientific (2 mentions)
Interleukin 6 (il 6), by Selleck Chemicals (1 mentions)
Sfemii, by STEMCELL (1 mentions)
4d nucleofector system, by Lonza (1 mentions)
Rnasin plus, by Promega (1 mentions)
Stemregenin 1, by STEMCELL (1 mentions)
V4xp 3024, by Lonza (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Flt3 ligand, by Thermo Fisher Scientific (1 mentions)
Stemspan aof, by STEMCELL (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Facsmelody sorter, by BD (1 mentions)
G csf, by STEMCELL (1 mentions)
Stemregenin1 sr1, by STEMCELL (1 mentions)
4 protocols using um171
1
Generating MDSC-like cells from CD34+
2
Electroporation of CD34+ HSPCs
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Mobilized peripheral-blood-derived human CD34+ HSPCs were cultured and electroporated as previously described55 (link). In brief, cryopreserved cells were thawed and cultured in StemSpan SFEMII medium (StemCell, 09655) supplemented with 1% penicillin–streptomycin (10,000 U ml−1), 1% l -glutamine (200 mM), 125 ng ml−1 hSCF (Peprotech, 300-07), 125 ng ml−1 hFLT3L (Peprotech, 300-19), 62.5 ng ml−1 hTPO (Peprotech, 300-18), 0.75 μM StemRegenin-1 (SR1, StemCell, 72344) and 35 nM UM171 (Sellekchem, S7608) unless stated otherwise. Then, 48 h after thawing (unless stated otherwise), cells were collected and electroporated using the Lonza 4D-Nucleofector system by resuspending the cells in P3 solution (Lonza, V4XP-3024) supplemented with 100–250 nM base editor mRNA, 15–20 μM sgRNA (IDT) and 1.2 U μl−1 RNase inhibitor (Promega RNAsin Plus, N2611) or 1.5–3% (v/v) glycerol. After electroporation, cells were then counted and transplanted after 24 h for in vivo experiments, or cultured for an additional 5–7 days in the same medium described above at 0.5 M ml−1 for in vitro experiments.
3
Expansion of Human CD34+ Hematopoietic Stem Cells
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Human CD34+ HSPCs were cultured as previously described20 (link),21 (link). CD34+ HSPCs were purchased from AllCells and had been isolated from G-CSF-mobilized peripheral blood from healthy donors. CD34+ HSPCs were cultured at 2.5 × 105–5 × 105 cells/mL in StemSpan™-AOF (Stemcell) supplemented with stem cell factor (SCF) (100 ng/mL), thrombopoietin (TPO) (100 ng/mL), FLT3–ligand (100 ng/mL), IL-6 (100 ng/mL) (all Peprotech) and UM171 (35 nM) (Selleckchem). Cells were cultured at 37 °C, 5% CO2, and 5% O2.
4
Establishing Human AML Xenografts
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Human patient-derived AML xenografts (PDX) were thawed and cultured in StemSpan SFEMII medium (StemCell, 09655) supplemented with 1% penicillin–streptomycin (10,000 U ml−1), 1% l -glutamine (200 mM), 50 ng ml−1 hSCF (Peprotech, 300-07), 50 ng ml−1 hFLT3L (Peprotech, 300-19), 25 ng ml−1 hTPO (Peprotech, 300-18), 10 ng ml−1 IL-3, 10 ng ml−1 G-CSF, 0.75 μM StemRegenin-1 (SR1, StemCell, 72344), 35 nM UM171 (Sellekchem, S7608) and 10 μM PGE2. Cells were transduced with a third-generation LV vector expressing mNeonGreen under a hPGK promoter (titre, ~2×1010 TU ml−1) at an MOI of 100 and cultured overnight at 37 °C in a humidified incubator under 5% CO2. Cells were collected the next day and transplanted into 4–8-week-old NBSGW female mice by tail-vein injection. Engraftment was monitored by peripheral blood collection and FACS analysis. Mice were euthanized when PDX AML cells exceeded around 20% of total white blood cells. BM and spleen were collected and either vitally frozen or FACS-sorted (BD FACS Melody sorter) to isolate mNeonGreen+ cells and retransplanted into new NBSGW recipients to obtain fully transduced PDXs.
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