On day 0, before mixing the PBLs with moDCs or anti-CD3 anti-CD28 beads, 1 × 106 cells/ml of PBLs per donor were pelleted (432 rcf, 5 min) and resuspended in CellTrace Far Red staining solution (Invitrogen C34564, 1:1000 in PBS) and incubated for 20 min at 37 °C. Afterwards, to remove any free dye in the solution, RPMI-FCS-L-Glu-AA medium was added at a volume of 5 times the original staining volume. The cells were spun down again, and the pellet was resuspended at 1 × 105 cells/50 μl in RPMI medium with 10% FCS. Subsequently, the CellTrace labeled PBLs were mixed with the moDC or beads and cultured for 5 days in RPMI-HS-L-Glu-AA medium as described above.
Celltrace far red staining solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CellTrace Far Red staining solution is a fluorescent cell labeling agent designed for long-term cell tracking and proliferation analysis. It provides a bright, stable signal that can be detected in the far-red spectrum. The solution is suitable for a variety of cell types and applications.
Automatically generated - may contain errors
4 protocols using celltrace far red staining solution
1
CellTrace Labeling of PBLs for Activation Assay
2
CellTrace Labeling of PBLs for Activation Assay
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On day 0, before mixing the PBLs with moDCs or anti-CD3 anti-CD28 beads, 1 × 106 cells/ml of PBLs per donor were pelleted (432 rcf, 5 min) and resuspended in CellTrace Far Red staining solution (Invitrogen C34564, 1:1000 in PBS) and incubated for 20 min at 37 °C. Afterwards, to remove any free dye in the solution, RPMI-FCS-L-Glu-AA medium was added at a volume of 5 times the original staining volume. The cells were spun down again, and the pellet was resuspended at 1 × 105 cells/50 μl in RPMI medium with 10% FCS. Subsequently, the CellTrace labeled PBLs were mixed with the moDC or beads and cultured for 5 days in RPMI-HS-L-Glu-AA medium as described above.
3
Multiparametric Cell Death and Proliferation Assay
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Apoptotic cells were stained using ApoTracker Green (BioLegend). Proliferating cells were analyzed using the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. For dilution assay to detect cell division, isolated B cells were stained with CellTrace Far-red staining solution (Thermo Fisher Scientific) according to the manufacturer’s instructions and cultured as described above. After 48 h, cells were harvested and stained with ApoTracker Green. Data were obtained using CytoFlex (Beckman Coulter) and analyzed using the FlowJo software 10.6.2 (BD Biosciences).
4
Microparticle and Cell Separation in Ferrofluid
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To facilitate optical microscope observation of the microparticle movement in the microfluidic channel, we used 10 µm polystyrene green fluorescent microparticles (1.05 g/mL density, Spherotech, USA) and 16 µm polystyrene red fluorescent microparticles (1.05 g/mL density, Spherotech, USA). For validation of the separation of the microfluidic channels, the green and red fluorescent polystyrene microparticle mixture was injected into the PEO mixed ferrofluid solution. In addition, 2–5 µm neural stem cells (NSCs) and 10–15 µm metastatic U87MG human glioblastoma cancer cells were used instead of the microparticles. CellTrace 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) staining solution (Thermo Fisher Scientific Inc., NY, USA) was used for U87MG glioblastoma cancer cells and CellTrace Far red staining solution (Thermo Fisher Scientific Inc., NY, USA) was used for NSC staining. The cells were gently re-suspended and were subsequently incubated at 37 °C for 20 minutes. The staining solution was removed by the centrifuge and the suspended cells were then injected 1 × 106 cells/mL suspension in medium into the microfluidic device. The fluorescent intensity was obtained from Image J software (National Institute of Health, USA) and the extracted data was analyzed using Matlab R2016a (Mathworks, USA).
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