Transfected BECs were left unstained or were labeled with CellTrackerTM Blue CMF2HC or Orange CMTMR (Thermo Fisher). BEC spheroids were generated in methyl cellulose over-night and seeded in methyl cellulose mixed with rat tail collagen (ratio 1:1) and 10% FBS. Where indicated, VEGF was added at 30 ng/ml. Sprout formation was evaluated after 18 h using an Axiovert 40 CFL microscope (Carl Zeiss AG) or by live cell imaging with a Cell-IQ® MLF system (CM Technologies, Tampere, Finland).
Orange cmtmr
Manufactured by Thermo Fisher Scientific
Orange CMTMR is a fluorescent dye used in cell biology applications. It is a derivative of the cell-permeant dye 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR). The dye can be used to label and track cells by covalently binding to intracellular components.
Automatically generated - may contain errors
Lab products found in correlation
Celltracker blue cmf2hc, by Thermo Fisher Scientific (1 mentions)
Axiovert 40 cfl microscope, by Zeiss (1 mentions)
Celltracker green cmfda, by Thermo Fisher Scientific (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Glutathione (gsh), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Cucl2 2h2o, by Merck Group (1 mentions)
Cgmp enzyme immunoassay kit, by Merck Group (1 mentions)
Dmem f12 medium, by GE Healthcare (1 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
Ckx53, by Olympus (1 mentions)
4 protocols using orange cmtmr
1
BEC Spheroid Formation and Sprouting Assay
2
Hydrogel-Mediated Cytoskeletal Manipulation
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DOTA was purchased from Shanghai Aladdin Bio‐Chem Technology Co., Ltd. PAa, HA (molecular weight: 1–2 × 105 Da), CuCl2·2H2O, Tris‐HCl, GSNO, GSH, cGMP Enzyme Immunoassay Kit, glutaraldehyde, MES, NHS, and EDC were purchased from Sigma‐Aldrich. DMEM‐F12 medium and fetal bovine serum (FBS) were from GE Healthcare Life Sciences. Cell Tracker Green CMFDA and Orange CMTMR were bought from Thermo Fisher Scientific. CCK‐8 was purchased from Dojindo.
3
Coculture of HUVECs and HUASMCs
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HUVECs and HUASMCs were prelabeled with CellTrackerTM Green CMFDA (Cat. No.: C2925, Thermo Fisher Scientific) and Orange CMTMR (Cat. No.: C2927, Thermo Fisher Scientific), respectively, according to the protocols provided by the vendor. Subsequently, the cells were detached, deposited, and then resuspeneded in their own growth medium supplemented with GSNO (10 μM) and GSH (30 μM). The two types of cells were diluted to the same density of 5000 cells mL−1, mixed at the ratio of 1:1, and then seeded onto the substrates placed in the 12-well plates at the density of 10,000 cells cm−2. After incubation for 3 h, the cells were gently washed with PBS and then photographed using an inverted fluorescence microscope (CKX53, Olympus, Japan; software: Olympus CellSens 2.3).
4
Tracking ADMSC Transplantation in Crushed Sciatic Nerve
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ADMSCs were isolated from two male rats. Immediately after crush, an intravascular injection of 200 µL of media containing 3 × 106 (link) NPC-labeled ADMSC was done through the sacra media artery using a 21-gauge hypodermic needle.
In parallel, as a positive control for fluorescent cells, a group of animals received a transplant of ADMSC labeled with the cell tracker orange CMTMR (5-(and 6)-(((4- chloromethyl) benzoyl) amino) tetramethyl-rhodamine) (Thermo Fisher Scientific Inc). For this purpose, the pellet containing the cells was suspended in α-Modified Eagle Medium (MEM) (Thermo Fisher Scientific) with the cell tracker and incubated for 45 minutes at 37°C under sterile conditions. Then the probe solution was replaced with fresh medium and the cells were incubated for 30 minutes. Two animals were used for each group of study: CMTMR, NPC, Sham, in total it was six animals.
Seven days post-crush, the animals were perfused as described previously by Usach et al.22 (link) The presence of NPC-and CMTMR-labeled ADMSC in the control and crushed sciatic nerves was evaluated in cryostat sections using fluorescence microscopy (Axio Vert.A1; Carls Zeiss).
In parallel, as a positive control for fluorescent cells, a group of animals received a transplant of ADMSC labeled with the cell tracker orange CMTMR (5-(and 6)-(((4- chloromethyl) benzoyl) amino) tetramethyl-rhodamine) (Thermo Fisher Scientific Inc). For this purpose, the pellet containing the cells was suspended in α-Modified Eagle Medium (MEM) (Thermo Fisher Scientific) with the cell tracker and incubated for 45 minutes at 37°C under sterile conditions. Then the probe solution was replaced with fresh medium and the cells were incubated for 30 minutes. Two animals were used for each group of study: CMTMR, NPC, Sham, in total it was six animals.
Seven days post-crush, the animals were perfused as described previously by Usach et al.22 (link) The presence of NPC-and CMTMR-labeled ADMSC in the control and crushed sciatic nerves was evaluated in cryostat sections using fluorescence microscopy (Axio Vert.A1; Carls Zeiss).
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