rDPCs were isolated from the incisor dental pulp tissues of 5-week-old male Wistar-ST rats (Shimizu Laboratory Supplies, Kyoto, Japan). All animal experiments were approved by the Animal Research Committee of Osaka Dental University and performed strictly according to the guidelines (Approval No. 21-02012; approval date: 23 March 2021). The obtained tissues were incubated with collagenase type I (3 mg/mL: Wako Pure Chemical Industries, Osaka, Japan) at 37 °C for 40 min. The fluid containing the cells was centrifuged for 3 min (1000× g). The cells were cultured in a Minimum Essential Medium Eagle-Alpha Modification (Nacalai Tesque, Kyoto, Japan), containing 20% fetal bovine serum and 1% penicillin-streptomycin solution (designated as culture medium), at 37 °C in a humidified atmosphere with 5% CO2. The fourth passage cells were used for this study. To characterize the immunophenotype of rDPCs, the antigen normally expressed on stem cells (APC anti-CD90, PE anti-CD44) and hemopoietic stem cells (PE anti-CD34) were selected. The cells were analyzed by flow cytometry using the FACSVerseTM system (BD, Franklin Lakes, NJ, USA).
Facsversetm system
Manufactured by BD
Sourced in United States
The FACSVerseTM system is a flow cytometry instrument designed for multi-parameter analysis and cell sorting. It features a compact design and supports a range of fluorescent probes for detecting and analyzing various cell types and populations. The instrument provides automated sample handling and advanced data analysis capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Binding buffer, by BD (2 mentions)
Collagenase type 1, by Fujifilm (1 mentions)
Anti cd8 fitc, by BioLegend (1 mentions)
Anti cd45 bv421, by BD (1 mentions)
Anti cd4 fitc, by BD (1 mentions)
Pi rnase staining buffer, by BD (1 mentions)
Apc conjugated annexin 5, by BD (1 mentions)
Annexin 5, by BD (1 mentions)
6 protocols using facsversetm system
1
Isolation and Characterization of Rat Dental Pulp Stem Cells
2
Flow Cytometry Analysis of Tumor-Infiltrating Immune Cells
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Tumor-infiltrating immune cells and splenocytes were analyzed using flow cytometry. Anti-CD45-BV421 (BD bioscience), anti-CD8-FITC (BioLegend), and anti-CD4-FITC (BD Bioscience) antibodies were used to characterize the cells (1×106) using the FACSVerseTM system (BD Bioscience, Piscataway, NJ, USA) in accordance with the manufacturer’s protocol.
3
Melarsomine's Effect on Cell Cycle
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To investigate the effect of melarsomine on the cell cycle, Abrams and D17 cells were
cultured with different concentrations (0, 80, and 120 µM) of melarsomine
in 2% FBS/DMEM for 48 hr. Thereafter, cells were harvested, washed, fixed with 70% cold
ethanol at −20°C for 2 hr, and washed twice with PBS. The cells were resuspended with
propidium iodide (PI)/RNase staining buffer (BD Biosciences, San Jose, CA, U.S.A.) and
incubated for 15 min at room temperature to stain DNA. The samples were analyzed by flow
cytometry using a FACSVerseTM system (BD Biosciences).
cultured with different concentrations (0, 80, and 120 µM) of melarsomine
in 2% FBS/DMEM for 48 hr. Thereafter, cells were harvested, washed, fixed with 70% cold
ethanol at −20°C for 2 hr, and washed twice with PBS. The cells were resuspended with
propidium iodide (PI)/RNase staining buffer (BD Biosciences, San Jose, CA, U.S.A.) and
incubated for 15 min at room temperature to stain DNA. The samples were analyzed by flow
cytometry using a FACSVerseTM system (BD Biosciences).
4
Isolation and Immunophenotyping of Lymphocytes
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The red blood cell lysate was added into the blood sample according to the volume of 1:5, to lyse at room temperature for 15 min. The sample was centrifuged at 1000 rpm for 5 min. The precipitates were resuspended and centrifuged. After washing the precipitates with phosphate‐buffered saline three times, white precipitates are lymphocytes. Then, the lymphocyte precipitation was resuspended with buffer containing 2% fetal bovine serum, and divided into two parts. One part is added with negative control antibody and the other part is added with 5 μL FITC‐CD4 antibodies (Biolegend cat 300505), APC‐CD3 antibodies (Biolegend cat 300311, and PE‐CD8 antibodies (Biolegend cat 344705). The cells were gently mixed, incubated, and then detected by flow cytometry using BD FACSVerseTM System.
5
Multicolor Flow Cytometry Analysis
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Fluorescence-labeled anti-mouse CD45-FITC (103108), anti-mouse CD11b-APC-Cy7 (101226), anti-mouse F4/80-PE (123109), anti-mouse Ly6G-PerCP-Cy5.5 (127616),anti-mouse Ly6C-APC (128016), anti-mouse CD3-PE (100206), anti-mouse B220-APC (103212) antibodies were obtained from BioLegend (San Diego, CA). All antibodies were used at 1:100 dilutions. For analysis of leukocytes from euthanized mice, peripheral blood cells were collected through cardiac puncture, followed by elimination of red blood cells by using a lysis buffer (Tiangen Biotech Inc, Beijing, China). The cells were also isolated from the colons as described above. cLP cells were incubated in FACS buffer with indicated antibodies for 30 min at 4 °C in the dark. After the removal of unbound antibodies, BD FACSVerseTM System (BD Biosciences, San Diego, CA) and FlowJo software were used for data acquisition and analysis.
6
Annexin V Apoptosis Assay for Transplanted Cells
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For Annexin V apoptosis assay, harvested lungs and kidneys were digested into single cell suspensions, washed 5 times with ice-cold PBS and then resuspended in 1× binding buffer (BD Biosciences, USA) at a concentration of 1 × 10 6 cells/mL. 100 μL of the cell suspension, 5 μL of APC-conjugated Annexin V (BD Biosciences, USA) and 5 μL of PI (BD Biosciences, USA) were sequentially added to a FACS tube followed by gently shaking and incubation for 15 min at room temperature in the dark. Following incubation, 400 μL of 1× binding buffer was added to each tube and cells were analyzed on a BD FACS VerseTM system within 1 hour. Gate was defined to remove debris and doublet cells using FSC and SSC.
Transplanted human cells were identified by detection of GFP signaling. Live and dead GFP positive cells were separated by APC-conjugated Annexin V and PI.
Transplanted human cells were identified by detection of GFP signaling. Live and dead GFP positive cells were separated by APC-conjugated Annexin V and PI.
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