This study was approved by the Ethics Committee of the General Hospital of Ningxia Medical University, China. Human umbilical cord-MSCs (UC-MSCs) were isolated from the umbilical cord connective tissue collected from healthy patients after obtaining informed consent. 3×105 UM-MSCs were seeded in 100mm culture plate containing Ultra Culture Serum-free Medium (Lonza, Switzerland) supplemented with 2% Pall Ultroser G Serum Substitute (Pall, USA), and cultured in the CO2 incubator maintained at 37°C with 5% CO2 and a humidified atmosphere. Medium was changed every 3 days. At approximately 90% confluence, cells were passaged and 3×105 UM-MSCs reseeded in a new 100mm culture plate. 1×106 UC-MSCs at passages 4 were seeded in a new 100mm culture plate. At 60–70% confluence, the medium was changed and the cells cultured for a further 24 h before harvesting the conditioned medium (CM). The CM was filtered (0.22-µm pore size) to remove cellular debris and concentrated using ultrafiltration units with a 3-kDa molecular-weight cutoff (Millipore, Burlington, MA) to obtain the MSC-CM.
Ultrafiltration units
Manufactured by Merck Group
Sourced in United States
Ultrafiltration units are laboratory equipment used for the separation and concentration of macromolecules, such as proteins, enzymes, and other biomolecules, from complex solutions. These units employ a semi-permeable membrane to selectively allow the passage of smaller molecules while retaining the larger target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Ultra culture serum free medium, by Lonza (2 mentions)
Quantity one software 4, by Bio-Rad (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 rslcnano hplc system, by Thermo Fisher Scientific (1 mentions)
Gemini c18 reverse phase column, by Phenomenex (1 mentions)
Kinetex c18 column, by Phenomenex (1 mentions)
Flavourzyme, by Merck Group (1 mentions)
Alcalase, by Merck Group (1 mentions)
0.2 m filter, by Merck Group (1 mentions)
9 protocols using ultrafiltration units
1
Isolation and Characterization of UC-MSCs
2
Conditioned Media Proteomic Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the preparation of the CM, 1×106 T24MshPFN1 or T24MshSCR cells were cultured until 80% confluent, and the media were replaced with DMEM containing 0.5% (v/v) FBS to prevent protein aggregation. The cells were cultured for a further 24 hours and the CM was collected and concentrated approximately 10-fold using ultra filtration units with a 3-kDa cut-off (Millipore Ltd. Ltd.), and analyzed for specific proteins using proteome profiler arrays for angiogenesis growth factors (Catalog #ARY007, R&D Systems Inc., Minneapolis, USA) according to the manufacturer's instructions. Quantitation of the detected spots was performed using the Quantity One Software 4.4.1 (BioRad Laboratories Inc., Amersham, England). Two replicates per sample were analyzed. Results for the different growth factors assayed are expressed as means±SD and statistical analysis was performed using Student's t test.
3
Generation and Concentration of Conditioned Media
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the generation of CM, the abovementioned cells were cultured for 24 hours, washed thoroughly, and cultured in 2 ml of DMEM supplemented with 2% fetal bovine serum and 2 mmol/l L-glutamine (Gibco). The CM was collected 24 hours later and concentrated 25-fold using ultrafiltration units with a 3 kDa cutoff (Millipore, Bedford, MA, USA). The concentrated CM was immediately cryopreserved at −80°C until use. MSC-CM, H-CM, and MSC-H-CM were derived from MSCs, hepatocytes, and a coculture of MSCs and hepatocytes, respectively. The control medium (non-CM or NCM) consisted of a similar medium without conditioning by human MSCs or hepatocytes.
4
Ultrafiltration of Conditioned Growth Medium
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Conditioned growth medium was concentrated 25-fold through ultrafiltration units (Millipore, Bedford, MA) with a 3-kDa cutoff [14 (link)]. The details are placed in Additional file 1 .
5
Encapsulation Efficiency of Oils in Nanoparticles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The efficiency of encapsulation of the oils in the nanoparticles was evaluated by the ultrafiltration/centrifugation method, using Millipore ultrafiltration units with exclusion pore size of 10 kDa. The concentrations of unencapsulated CVC and LNL in the filtrate were determined by HPLC, and the encapsulation efficiency was calculated as the difference between the initial concentration added (100%) and the concentration in the ultrafiltrate. The analyses (in triplicate) were performed with an UltiMate 3000 RSLCnano HPLC system (Thermo Scientific). For CVC, a Phenomenex Gemini C18 reverse phase column (100 × 4.6 mm; 2.6 μm) was used, and the mobile phase consisted of acetonitrile:water (50:50, v/v), at a flow rate of 1 mL/min. For LNL, a Phenomenex Kinetex C18 column (250 × 4.6 mm; 3 μm) was employed, and the mobile phase consisted of acetonitrile:water (65:35, v/v), at a flow rate of 1.5 mL/min. The analytical curves used for quantification could be described by the following equations:
6
Chia seed protein extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Alcalase (E.C. 3.4.21.62) and flavourzyme (E.C. 232-752-2) were acquired from Sigma Aldrich (St. Luis, MO, USA). Ultrafiltration units were purchased from Millipore (Bedford, MA, USA). Chia (Salvia hispanica L.) seeds were obtained from Healthworks (pesticide-free, Scottsdale, AZ, USA). All chemical reagents used in this study were analytical grade or HPLC grade.
7
Isolation and Characterization of UC-MSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
This study was approved by the Ethics Committee of the General Hospital of Ningxia Medical University, China. Human umbilical cord-MSCs (UC-MSCs) were isolated from the umbilical cord connective tissue collected from healthy patients after obtaining informed consent. 3×10 5 UM-MSCs were seeded in 100mm culture plate containing Ultra Culture Serum-free Medium (Lonza, Switzerland) supplemented with 2% Pall Ultroser G Serum Substitute (Pall, USA), and cultured in the CO2 incubator maintained at 37°C with 5% CO2 and a humidified atmosphere. Medium was changed every 3 days. At approximately 90% confluence, cells were passaged and 3×10 5 UM-MSCs reseeded in a new 100mm culture plate. 1×10 6 UC-MSCs at passages 4 were seeded in a new 100mm culture plate. At 60%-70% confluence, the medium was changed and the cells cultured for a further 24 h before harvesting the conditioned medium (CM). The CM was filtered (0.22-μm pore size) to remove cellular debris and concentrated using ultrafiltration units with a 3-kDa molecular-weight cutoff (Millipore, Burlington, MA) to obtain the MSC-CM.
8
Flagellin-Activated ADSC Secretome Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
When reaching 80–90% confluency, the culture media was changed to a mixture of D10 media and flagellin (100 ng/ml). After 48 h incubation at 37°C, flagellin-activated ADSCs were washed three times with warm PBS and re-supplemented with serum-free DMEM. After 48 h, the CM was collected, filtered with a 0.2-µm filter (EMD Millipore) and concentrated 50-fold using ultrafiltration units with a 3-kDa molecular weight cutoff (EMD Millipore) (26 (link)). The concentrated F-CM was stored at −80°C before being used for the following experiments. The CM was prepared using the same method, except no flagellin was added.
9
Metabolomic Analysis of Cells and Medium
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples of cells and of conditioned medium were obtained 96 h after administration of FF/CAP18. Cell samples were washed twice with a 5% solution of mannitol and covered with methanol. Cells were harvested after addition of the internal standard solution (Human Metabolome Technologies, Tsuruoka, Japan), and subjected to centrifugation for 5 min at 2,300 × g, 4°C. The aqueous layers were collected into ultrafiltration units (EMD Millipore, Billerica, MA, USA) and subjected to centrifugation for 2.5 h at 9,600 × g, 4°C. The conditioned medium from cell cultures was directly collected to prepare medium samples. The sampled medium was mixed with the internal standard solution, and subjected to centrifugation for 2.5 h at 9,600 × g, 4°C.
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!