ADSCs cultured in FBS containing media, DMEM (HyClone) supplemented with 10% FBS (HyClone), SFM (CellCor, Xcell Therapeutics). StemPro MSC SFM XenoFree (Thermo Fisher Scientific), or MesenCult-ACF Plus Culture Kit (STEMCELL Technologies) were compared. ADSCs were seeded at 4 × 103 cells/cm2 in T25 flasks (Corning) and passaged every 4 days, except every 3 days for SFM. Flask of FBS containing media and SFM group did not need to be pre-coated. When using StemPro MSC SFM XenoFree and the MesenCult-ACF Plus Culture Kit, the flasks were pre-coated with CELLstart Substrate (Thermo Fisher Scientific) and Animal Component-Free Cell Attachment Substrate (STEMCELL Technologies), respectively. At each passage, ADSCs were detached and counted using a NucleoCounter NC-250 (ChemoMetec, Allerod, Denmark) and seeded in new T25 flasks. The population doubling time (PDT) was determined at each passage by the formula: PDT = culture period in hours / {log (number of cells T0) – log (number of cells T1)} / log(2), where T0 is the seeding cell number and T1 is the cell number at harvest. The accumulation cell number (ACN) was determined at each passage by using formula: ACN = (ACN of previous passage / T0) x T1.
Stempro msc sfm xenofree
Manufactured by Thermo Fisher Scientific
Sourced in United States
StemPro® MSC SFM XenoFree is a serum-free and xeno-free medium designed for the culture of human mesenchymal stem cells (MSCs). The core function of this product is to provide a defined, animal component-free culture environment for the expansion of MSCs.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Cellbind surface cell culture plates, by Corning (1 mentions)
Raw 264, by Korean Cell Line Bank (1 mentions)
Cellstart substrate, by Thermo Fisher Scientific (1 mentions)
Mesencult acf plus culture kit, by STEMCELL (1 mentions)
Animal component free cell attachment substrate, by STEMCELL (1 mentions)
T25 flask, by Corning (1 mentions)
Nucleocounter nc 250, by ChemoMetec (1 mentions)
Poietics human mscs, by Lonza (1 mentions)
4 protocols using stempro msc sfm xenofree
1
Comparative Evaluation of ADSC Culture Conditions
2
Culturing Normoxic and Hypoxic MSCs
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Poietics human MSCs (passage 2) were purchased from Lonza (Basel, Switzerland), expanded until passage 4, and used as normoxic MSCs (norMSCs). The cells were cultured and passaged with StemPro MSC SFM XenoFree (Thermo Fisher Scientific, Waltham, MA, USA) under normal oxygen conditions (21% O2) and 5% CO2 at 37 °C. Passage 4 cells were frozen and stored using liquid nitrogen. StemPro BM MSCs (passage 4; Thermo Fisher Scientific) were purchased from Thermo Fisher Scientific and used as hypoxic MSCs (hypoMSCs). The cells were cultured in StemPro MSC SFM XenoFree (Thermo Fisher Scientific) under hypoxic conditions (5% O2) and 5% CO2 at 37 °C. Cryopreservation solution with 10% DMSO was used for cryopreservation of these cells.
3
Stem Cell Media Comparison Protocol
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Three different media were used: StemPro–, composed of StemPro® MSC SFM XenoFree (Gibco™, Thermo Fisher, www.thermofisher.com ) supplemented with 2 mM l -glutamine (Gibco™), and 100 U/mL penicillin and 0.1 mg/mL streptomycin (Gibco™); StemPro+, (StemPro– supplemented with StemPro supplement); and StemPro E8 (StemPro– supplemented with Essential 8™ Medium supplement (Gibco™)).
4
Immunomodulation by rhBMP-2-primed Stem Cells
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Human ES-MSCs were provided by Daewoong Pharmaceutical Co., Ltd. ES-MSCs were cultured in a StemPro® MSC SFM XenoFree (Gibco, Waltham, MA, USA) supplemented with 1% L-glutamine (Gibco) and 1% penicillin/streptomycin on the Corning® CellBIND® surface cell culture plates (Corning Inc., Corning, NY, USA) at 37°C. For priming with rhBMP-2, cells were treated with rhBMP-2 (100 ng/ml) for 24 h. All experiments were performed using ES-MSCs at passage number 12. RAW264.7, murine macrophage cell line, was purchased from Korean Cell Line Bank (KCLB, Seoul, Korea). RAW264.7 cells were grown in RPMI-1640 medium at 37°C. To determine immunomodulation effect by paracrine factors from rhBMP-2-primed ES-MSCs in RAW264.7, cells were seeded in 60 mm culture dish at confluency 70%–80%. RAW264.7 cells were incubated with conditioned medium (CM) from ES-MSCs/rhBMP-2-primed ES-MSCs mixed with RPMI1640 (2:1 ratio). After 2 h, LPS was added for 6 h. Cells were lysed with RIPA buffer for western blotting analysis, and CM was used for ELISA.
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