The cell apoptosis was detected using an Annexin V-FITC/PI apoptosis detection kit, and analyzed by a BD FACSCelesta ow cytometer (BD Biosciences, San Diego, CA). For intracellular ROS level measurement, cells were stained with 10 µM dichloro-dihydro-uorescein diacetate (DCFH-DA) dye in serum-free culture medium at 37 °C for 20 min in the dark and washed with PBS, and the relative DCFH uorescence of the cells was analyzed with a BD FACSCelesta ow cytometer.
Facscelesta ow cytometer
Manufactured by BD
The BD FACSCelesta flow cytometer is a high-performance instrument designed for multicolor flow cytometry analysis. It uses advanced optical and fluidic technologies to enable rapid and accurate detection and analysis of cells or particles in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Viafluor 405, by Biotium (1 mentions)
Viafluor 405, by FlowJo (1 mentions)
Annexin 5 apc, by BioLegend (1 mentions)
Facscelesta flow cytometer, by BD (1 mentions)
Annexin 5 binding buffer, by BioLegend (1 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Anti cd3, by BioLegend (1 mentions)
Anti cd28, by BioLegend (1 mentions)
Cfse cell division tracker kit, by BioLegend (1 mentions)
5 protocols using facscelesta ow cytometer
1
Apoptosis and ROS Detection
2
Apoptosis and ROS Detection
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The cell apoptosis was detected using an Annexin V-FITC/PI apoptosis detection kit, and analyzed by a BD FACSCelesta ow cytometer (BD Biosciences, San Diego, CA). For intracellular ROS level measurement, cells were stained with 10 µM dichloro-dihydro-uorescein diacetate (DCFH-DA) dye in serum-free culture medium at 37 °C for 20 min in the dark and washed with PBS, and the relative DCFH uorescence of the cells was analyzed with a BD FACSCelesta ow cytometer.
3
Tracking GMSC Homing in SCID Mice
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GMSC were labeled with ViaFluor 405 (Biotium, Fremont, CA) and then injected intraperitoneally into female SCID mice that had received one implant containing cartilage and RASF 3 days earlier. Labeled GMSC were maintained in culture for the duration of the experiment. The implants were harvested at 4 days and the cartilage was isolated and digested in collagenase to release adherent cells. The cell suspension was analyzed on a BD FACSCelesta ow cytometer (BD, Franklin Lakes, NJ) and data were analyzed by using FlowJo v10.8.1 (Ashland, OR).
The cultured ViaFluor 405 labeled GMSC were acquired on the cytometer on days 1, 2, 3, and 4 to track the fluorescence intensity of the dye over the course of the experiment to identify a positive cell gating strategy for the cells harvested from the implants on Day 4.
The cultured ViaFluor 405 labeled GMSC were acquired on the cytometer on days 1, 2, 3, and 4 to track the fluorescence intensity of the dye over the course of the experiment to identify a positive cell gating strategy for the cells harvested from the implants on Day 4.
4
Co-culture Assay for RASF and GMSC
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RASF were labeled with ViaFluor 405 according to the manufacturers’ protocol and then 5,000 RASF were plated per well in a 24 well plate. GMSC were labeled with CFSE and then either 5,000 or 500 GMSC were added to the appropriate wells of the 24 well plate. The cells were allowed to co-incubate for 3 days and were trypsinized, resuspended in AnnexinV binding buffer, stained with AnnexinV-APC (Biolegend, San Diego, CA) and then analyzed on a BD FACSCelesta flow cytometer. For the cell crosstalk experiment, the RASF were separately labeled with ViaFluor 405 and the GMSC were labeled with ViaFluor 488. The cells were washed extensively and incubated in complete media in suspension for 1 hr. to ensure no residual dye reactivity was present. The cells were then added to a 24 well plate at the indicated ratios and cultured for two days. For the transwell incubation, the RASF were added to the bottom well and the GMSC were added to the top transwell chamber according to the manufacturer’s directions. The cells were cultured for 2 days and were then collected and analyzed on a BD FACSCelesta ow cytometer.
5
Immunomodulatory Effects of GMSC-Exo and GMSC
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To examine and compare the effects of GMSC-Exo and GMSC on the proliferation of CD4 + T cells in vitro, we applied CFSE Cell Division Tracker Kit (Biolegend, San Diego, CA). Mouse CD4 + T cells were isolated from splenocytes by a CD4 + T Cell Isolation Kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer's protocol and labeled with CFSE, then cultured alone or with GMSC-Exo at the ratio of 10 ug/40 ug/90 ug: 10 6 (GMSC-Exo: CD4 + T cells), or with GMSC at the ratio of 1:1, 1:10 or 1:50 (GMSC:
CD4 + T cells). The anti-CD3 (5 ug/ml, Biolegend, San Diego, CA) was pre-coated on the bottom of culture plate and the anti-CD28 (5 ug/ml, Biolegend, San Diego, CA) was added with GMSC-Exo or GMSC. The GMSC-Exo/GMSC were added for 3 days, then the cells were analyzed using a BD FACSCelesta ow cytometer and the levels of Th1, Th17, and Treg cytokines (IFN-γ, IL-17A, and IL-10) were detected by ELISA according to the manufacturer's protocols [31, 32] .
CD4 + T cells). The anti-CD3 (5 ug/ml, Biolegend, San Diego, CA) was pre-coated on the bottom of culture plate and the anti-CD28 (5 ug/ml, Biolegend, San Diego, CA) was added with GMSC-Exo or GMSC. The GMSC-Exo/GMSC were added for 3 days, then the cells were analyzed using a BD FACSCelesta ow cytometer and the levels of Th1, Th17, and Treg cytokines (IFN-γ, IL-17A, and IL-10) were detected by ELISA according to the manufacturer's protocols [31, 32] .
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