Single-cell suspensions were prepared from hNTSCs and hBM-MSCs. Cells were incubated for 30 min at 4 °C with stage-specific embryonicantigen-3 (SSEA-3) antibody (1:20, Abcam) followed by Alexa Fluor 488 anti-rat antibody (1:200, Thermo Fisher Scientific). After incubation with the SSEA-3 antibody, the cells were incubated with CD105 antibody (1:50, PE-conjugated, BD Biosciences) for 30 min at 4 °C for double staining. The cells were resuspended in fluorescence-activated cell sorting (FACS) buffer and acquired through FACS Canto II (BD Biosciences) with DIVA software.
Alexa fluor 488 anti rat antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa Fluor 488 anti-rat antibody is a fluorescently labeled secondary antibody that specifically binds to rat primary antibodies. It is designed for use in various immunoassay techniques, such as flow cytometry, immunofluorescence microscopy, and Western blotting, to detect and visualize target proteins in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (1 mentions)
Ssea 3 antibody, by Abcam (1 mentions)
Cd105 antibody, by BD (1 mentions)
Axio imager, by Zeiss (1 mentions)
Ly6g ly6c gr 1 clone rb6 8c5, by BioLegend (1 mentions)
Alexa fluor 647 anti rat antibody, by Thermo Fisher Scientific (1 mentions)
Cd11b, by Abcam (1 mentions)
4 protocols using alexa fluor 488 anti rat antibody
1
Immunophenotyping of hNTSCs and hBM-MSCs
2
Quantifying Vascular Endothelial Cadherin Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cross-sections (thickness: 10 µm) of the cryopreserved thoracic aortas from C57BL/6J mice were used in immunofluorescence microscopy experiments. Cryosections were fixed in 4% paraformaldehyde for 15 min at room temperature. Non-specific binding was blocked with 10% goat serum in PBS. Sections were incubated overnight at 4 °C with a rat anti–mouse VE-cadherin primary antibody diluted at 1:50 (Clone 11D4.1 recognizing the extracellular domain of VE-cadherin, reference 550548, BD Biosciences, Franklin Lakes, NJ, USA). The slides were then incubated for 1-h with a secondary Alexa Fluor 488 anti-rat antibody diluted at 1:500 (references A-11006, Thermo Scientific, Waltham, MA, USA). Antibodies were diluted in PBS including 5% goat serum. Cell nuclei were stained with DAPI. One section from each aorta was observed by using a fluorescence microscope (Axio Imager, Zeiss, Oberkocheņ, Germany) and fluorescence was quantified using the software ImageJ after subtracting the autofluorescence of elastin and the background.
3
Multiparametric Profiling of Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary antibodies used: CD68 (clone FA-11, eBioscience, catalog no.: 14-0681-82); MHC Class II (I-A/I-E) Monoclonal Antibody, eFluor 450, (clone M5/114.15.2, eBioscience, catalog no.: 12-5321-82); phycoerythrin conjugated anti-mouse Ly6G/Ly6C (GR-1) (clone RB6-8C5, Biolegend, catalog no.: 108408); CD4 (Abcam, catalog no.: EPR19514); CD11b (Abcam, catalog no.: EPR1344); CD45R (clone RA3-6B2, Abcam, catalog no.: ab64100); S100A9 (clone 2B10, Abcam, catalog no.:ab105472); CD3e (Abcam, catalog no.: ab5690).
Secondary antibodies used: Alexa Fluor 488 anti-rat antibody (Life Technologies, catalog no: A21208); Alexa Fluor 647 anti-rat antibody (Life Technologies, catalog no.: A2124); Alexa Fluor 647 anti-rabbit antibody (Life Technologies, catalog no.: A31573).
Secondary antibodies used: Alexa Fluor 488 anti-rat antibody (Life Technologies, catalog no: A21208); Alexa Fluor 647 anti-rat antibody (Life Technologies, catalog no.: A2124); Alexa Fluor 647 anti-rabbit antibody (Life Technologies, catalog no.: A31573).
4
Cell Injury and Surface LAMP1 Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Changes were monitored as described previously.33 Cells cultured on coverslips were injured as described above by rolling of glass beads in the presence of lysine-fixable TRITC-dextran. After allowing repair to occur for 5 min at 37 °C and blocking with 3% BSA (in CIM) for 10 min at 4 °C, cell-surface LAMP1 was immunolabeled for 30 min at 4 °C using antibody specific for the luminal domain of LAMP1 (Santa Cruz) in blocking solution, followed by labeling with Alexa Fluor 488-anti-rat antibody (Life Technologies) for 15 min at 4 °C. Cells fixed in 4% PFA were stained with Hoechst 33342 and imaged as described in Supplementary Methods. The increase in cell-surface LAMP1 staining intensity of individual injured (TRITC-positive) cells was normalized to the average cell-surface LAMP1 staining intensity of all (>125) uninjured (TRITC-negative) cells. The percentage increase in cell-surface LAMP1 staining for each injured cell was then determined (divided by the average value for the uninjured cells). This percentage increase was then averaged for all the injured cells (>400 measured).
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!