Lentivirus with green fluorescent protein (GFP) was purchased from (GKN, China, product No.: LVCON313). The 3rd generation hUC-MSCs were seeded at a density of 1 × 105/well and incubated in 6-well plates 18–24 hours before transfection. The next day, lentiviruses with GFP fluorescent markers were transfected. The number of cells at the time of transfection was approximately 3×105/well. The multiplicity of infection (MOI) was 20. The medium was changed after 24 hours of co-culture. Fluorescence was observed with an inverted fluorescence microscope. Based on the fluorescence intensity, cells that failed to stably express GFP were screened with 4 μg/mL of puromycin. After the stable expression of GFP was established, we examined whether cell proliferation and apoptosis were affected before and after transfection using a cell counting kit-8 (CCK-8) and apoptosis kits (Annexin V-APC and 7-AAD, Elabscience, China). Ultra-low adsorption round-bottom 96-well culture plates (Corning 7007, USA) were sterilized with UV light prior to the spheroidization of hUC-MSCs. Then, 100 μL of hUC-MSCs suspension at a concentration of 2.5×106/mL was added to each well and the cells were gently shaken to concentrate them at the bottom of the plate. After 48 hours of incubation, hUC-MSCs spheres were formed and removed from the wells with a pipette.
7 aad
Manufactured by Elabscience
Sourced in China
7-AAD (7-aminoactinomycin D) is a fluorescent dye commonly used in flow cytometry applications. It binds to DNA and can be used to identify dead or dying cells in a sample.
Automatically generated - may contain errors
5 protocols using 7 aad
1
Lentiviral Transduction of hUC-MSCs for GFP Expression
2
CD106 Antibody Staining and 7AAD Viability
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Add the CD106 antibody (BD, USA) to the cells obtained in cells that did not use the Dead Cell Removal Kit, incubate them in the dark at 4°C for 30 min, and wash them twice. Then, the cells were added 1 μM 7AAD (Elabscience, China), incubated at room temperature for 15 min in the dark and washed twice. And set up the untreated control group (CON or CON+), which only carried out CD106 antibody incubation.
3
Apoptosis Detection by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After cell transfection, 106 cells from each group were washed and stained Annexin V-APC and 7AAD (Elabscience, Wu Han, China) at room temperature for 15 min. Flow cytometry (BD FACSCalibur, New Jersey, USA) was used to evaluate the proportion of apoptotic cells (low right corner of the flow cytometry graph regarded as apoptotic cells).
4
Apoptosis Evaluation of TmSm Proteins and ADM
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A549 cells, L-02 cells, and CSCs were resuspended into single cell suspension and plated at a density of 4 × 105 cells/well into six-well plates. After culturing for 24 h, A549 and L-02 cells were treated with TmSm proteins, while CSCs were incubated with TmSm proteins, ADM, and their combination, respectively. Following incubation with drugs for 24 h, cells were harvested by centrifugation at 2,000 rpm for 5 min and washed twice with PBS. Further, cells were resuspended in 100 µl binding buffer. Subsequently, 5 μl Annexin V-FITC and 5 μl propidium iodide (PI) (Elabscience, China) were added to each tube for 30 min incubation in the dark at 25°C to detect the effect of TmSm protein drugs on cell apoptosis. Meanwhile, 5 μl Annexin V-FITC and 5 μl 7-AAD (Elabscience, China) were used to detect the effect of ADM on cell apoptosis. The resultant samples were immediately analyzed by flow cytometry using FITC channel (excitation: 488 nm and emission: 525nm), PI channel (excitation: 535 nm and emission: 615 nm), and 7-AAD channel (excitation: 488 nm and emission: 670 nm).
5
Induced THP-1 Macrophage Phenotypic Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The induced THP-1 macrophages were collected, Fc receptors were blocked by human Fc Receptor Blocking Solution (Maokangbio), stained with FITC Anti-Mouse/Human CD11b Antibody (Elabscience, clone:M1/70), 7-AAD (Elabscience), APC Anti-Human CD206 Antibody (Elabscience, clone:15–2) and PE Anti-Human CD86 Antibody (Elabscience, clone:BU63), and detected and analyzed by Beckman cytoflex flow cytometry.
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!