We characterized the primary cultured cells from human adipose tissues. The cells were observed their morphology by inverted microscope and characterized using surface markers and fluorescence-activated cell sorting (FACS) analyses, with labeling by fluorescein-isothiocyanate (FITC)-conjugated monoclonal antibodies directed against the cluster of differentiation (CD) markers (BD Biosciences Pharmingen, San Diego, CA) CD73, CD90 or CD105 or with phycoerythrin (PE)-conjugated monoclonal antibodies directed against CD31, CD34 or CD45. Analysis was performed using a FACscan argon laser cytometer (Becton Dickinson, San Jose, CA). Additionally, the multipotency of the cells was determined using a mesenchymal differentiation kit (Invitrogen) as previously described [10 (link)]. The differentiation of hAT-MSCs into adipocytes, osteocytes and chondrocytes was confirmed by Oil Red O, Alizarin Red S and Alcian blue staining, respectively. All experiments were conducted in triplicate.
Facscan argon laser cytometer
Manufactured by BD
Sourced in United States
The FACScan argon laser cytometer is a flow cytometry instrument used for the analysis and sorting of cells. It utilizes an argon laser as the light source for excitation of fluorescent dyes or labels attached to cells. The FACScan is capable of detecting and measuring multiple parameters, such as size, granularity, and fluorescence intensity, on individual cells within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Fitc conjugated monoclonal antibodies, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions)
Cd34 fitc, by BD (1 mentions)
Cd45 apc, by BD (1 mentions)
Cd44 pe, by BD (1 mentions)
Mir 331 3p inhibitor, by GenePharma (1 mentions)
Mir nc, by GenePharma (1 mentions)
7 protocols using facscan argon laser cytometer
1
Characterization of Human Adipose-Derived Cells
2
Cell Cycle Analysis by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flow cytometry was done using a FACScan argon laser cytometer (Becton Dickinson, Franklin Lakes, NJ, USA), and the fraction of cells in each phase of the cell cycle was determined using the cell cycle analysis platform in the FlowJo software (Tree Star Inc., Ashland, OR, USA).
3
Adipose-Derived Stem Cell Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SVF consists of a heterogeneous population of cells including ADSC, endothelial cells, and endothelial precursor cells, whereas ADSC are a homogeneous population of one type of cells [22 (link)]. Adipose tissue was harvested from each rat's own paratesticular fat at the time of randomization (ADSC injection groups) or shortly before surgical procedures (SVF injection groups). SVF isolation and ADSC culture were performed as described previously in detail [23 (link)]. Flow cytometry to characterize SVF and ADSC was performed with a FACScan argon laser cytometer (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com ) as described previously in detail [23 (link)]. Differentiation of ADSC was confirmed as described previously in detail [23 (link)].
4
Isolation and Characterization of Rat ADSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A 12-week-old male SD rat was used to isolate ADSCs. General anesthesia was achieved by intraperitoneal injection of pentobarbital sodium (HWRK Chem Co., Ltd.). Primary cells were acquired as previously described.10 (link) Cells were cultured in Dulbecco's modified Eagle's medium/F-12 containing 1% antibiotic-antimycotic solution and 10% fetal bovine serum (Life Technologies, Carlsbad, CA, USA) at 37°C in 95% humidity and 5% CO2. The medium was changed every 2 days.
Cultured cells were analyzed by flow cytometry with a FACScan argon laser cytometer (BD Biosciences, San Jose, CA, USA) to determine whether the cells met the criteria of the joint statement of the International Federation for Adipose Therapeutics and Science and the International Society for Cellular Therapy.11 (link) Briefly, cultured passage 2 cells were harvested in 0.05% trypsin and washed with PBS (Thermo Fisher Scientific Inc.) and then protected from light and incubated for 30 min in flow cytometry buffer containing APC/Cy7-conjugated anti-rat CD29, APC-conjugated anti-rat CD45, PerCP-conjugated anti-rat CD90 (BioLegend, Inc., San Diego, CA, USA), and PE-Cy7-conjugated anti-rat CD31 (Thermo Fisher Scientific, Inc.). Separate tubes were prepared for each antibody to provide compensation controls. An extra sample tube containing unstained cells was used as a control.
Cultured cells were analyzed by flow cytometry with a FACScan argon laser cytometer (BD Biosciences, San Jose, CA, USA) to determine whether the cells met the criteria of the joint statement of the International Federation for Adipose Therapeutics and Science and the International Society for Cellular Therapy.11 (link) Briefly, cultured passage 2 cells were harvested in 0.05% trypsin and washed with PBS (Thermo Fisher Scientific Inc.) and then protected from light and incubated for 30 min in flow cytometry buffer containing APC/Cy7-conjugated anti-rat CD29, APC-conjugated anti-rat CD45, PerCP-conjugated anti-rat CD90 (BioLegend, Inc., San Diego, CA, USA), and PE-Cy7-conjugated anti-rat CD31 (Thermo Fisher Scientific, Inc.). Separate tubes were prepared for each antibody to provide compensation controls. An extra sample tube containing unstained cells was used as a control.
5
Isolation and Characterization of Adipose-Derived Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ADSC was cultured as previously described using paratesticular fat harvested from each rat (20 (link)). ADSC was cultured in the conventional 2D culture system until passage number three.
Flow cytometry was performed with a FACScan argon laser cytometer (BD Biosciences, San Jose, CA) to determine whether the processed lipoaspirate cells were characteristic of ADSC according to a previous study (21 (link)). Briefly, cells were harvested in 0.25% trypsin/EDTA and fixed for 30 minutes in ice-cold 2% for-maldehyde. The fixed ADSCs were washed in flow cytometry buffer and incubated for 30 minutes in flow cytometry buffer containing anti-CD29Pacific Blue, CD44PE, CD45APC, and CD34FITC (BD Biosciences). Differentiation of ADSC was confirmed as described previously (20 (link)).
Flow cytometry was performed with a FACScan argon laser cytometer (BD Biosciences, San Jose, CA) to determine whether the processed lipoaspirate cells were characteristic of ADSC according to a previous study (21 (link)). Briefly, cells were harvested in 0.25% trypsin/EDTA and fixed for 30 minutes in ice-cold 2% for-maldehyde. The fixed ADSCs were washed in flow cytometry buffer and incubated for 30 minutes in flow cytometry buffer containing anti-CD29Pacific Blue, CD44PE, CD45APC, and CD34FITC (BD Biosciences). Differentiation of ADSC was confirmed as described previously (20 (link)).
6
Cytometric Analysis of HBMSC Surface Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were analyzed by flow cytometry using antibodies described in Table 1 . HBMSCs were cultured in control medium 72 h before the analysis. Aliquots containing 5 × 105 cells were incubated with primary antibodies. Flow cytometry with a FAC scan argon laser cytometer (BD, Biosciences, San Jose, CA, USA) was performed according to a previous study [57 (link)]. Briefly, the cells were harvested in 0.25% trypsin/EDTA and fixed for 30 min in ice-cold 70% EtOH. The fixed cells were washed in flow cytometry buffer (PBS, 2% FBS, 0.2% Tween 20) and incubated for 30 min in flow cytometry buffer containing fluorescein isothiocyanate-conjugated monoclonal antibodies CD antigens CD29, CD31, CD34, CD44, CD45, CD49d, CD56, CD62e, CD90, CD105, CD106, CD133, and CD166 to check for specific stem cell surface markers. HBMSCs were stained with a phycoerythrin-conjugated nonspecific IgG to assess background fluorescence [67 (link)]. Cell surface marker expression was determined by comparison with an isotype control on a histogram plot.
7
Cell Cycle Analysis of Preadipocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Preadipocytes were seeded in 96-well plates and incubated for 24 h. By this time, the cells reached confluence and were transfected with either miR-331-3p mimic, miR-NC, or miR-331-3p inhibitor (GenePharma, Shanghai, China). Preadipocytes were harvested by digestion with 0.25% trypsin and washed 3 times with PBS to remove cell debris. The cells were then fixed in cold 70% ethanol overnight and treated with 1 mg/mL RNaseA and propidium iodide (PI) for 30 min at 37°C. The cells were then filtered through a 0.75 μm membrane prior to analysis on a FACScan argon laser cytometer (BD, USA).
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!