αvβ3 integrin expression in PANC-1, HeLa and 293T cells was determined using a BD FACS-Canto II cytofluorimeter (Becton-Dickinson Biosciences, Franklin Lakes, NJ, USA) by means of FITC-labeled mouse anti-human CD51/CD61 antibodies (BD Pharmingen, San Jose, CA, USA). 10,000 living cells were taken into account.
Facs canto 2 cytofluorimeter
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The BD FACS-Canto II is a flow cytometer that can analyze and sort cells based on their physical and fluorescent characteristics. It utilizes multiple lasers and detectors to measure various parameters of individual cells within a sample.
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8 protocols using facs canto 2 cytofluorimeter
1
αvβ3 Integrin Expression Analysis
2
Leiomyoma Cell Culture and αvβ3 Integrin Analysis
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Human kidney (293T) and human pancreatic (PANC-1) cell lines were obtained from the Cell Collection of the Institute of Cytology RAS (Saint-Petersburg, Russia). Primary leiomyoma cells were obtained after myomectomy in the D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology (Saint-Petersburg, Russia) as previously reported [43 (link)]. Briefly, dissected collagenase IV treated fibroids were resuspended in AmnioMax Basal Medium with 10% AmnioMax Supplement serum (Thermo Fisher Scientific, Carlsbad, CA, USA). The UL cells suspension was transferred to cultural flasks and after the first passage the AmnioMax was substituted by DMEM-F12 with 10% fetal bovine serum (Thermo Fisher Scientific, Carlsbad, CA, USA). The cell culturing was continued for up to 6 weeks at 37 °C with 5% CO2.
The expression of αvβ3 integrins in leiomyoma cells was determined by flow cytometry after cells detaching from flasks with 5 mM EDTA in 1× phosphate-buffered saline (PBS) (Rosmedbio, Saint-Petersburg, Russia) and staining with FITC mouse anti-human CD51/CD61 antibodies (BD Pharmingen, San Jose, CA, USA) for 20 min at room temperature [43 (link)]. Flow cytometry was conducted using BD FACS-Canto II cytofluorimeter (Becton-Dickinson Biosciences, Franklin Lakes, NJ, USA). A total of 10,000 cells were taken into account.
The expression of αvβ3 integrins in leiomyoma cells was determined by flow cytometry after cells detaching from flasks with 5 mM EDTA in 1× phosphate-buffered saline (PBS) (Rosmedbio, Saint-Petersburg, Russia) and staining with FITC mouse anti-human CD51/CD61 antibodies (BD Pharmingen, San Jose, CA, USA) for 20 min at room temperature [43 (link)]. Flow cytometry was conducted using BD FACS-Canto II cytofluorimeter (Becton-Dickinson Biosciences, Franklin Lakes, NJ, USA). A total of 10,000 cells were taken into account.
3
Transfection of PANC-1 Cells with RGD Peptide
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PANC-1 cells were seeded at a density of 5.0 × 104 cells per well in 48-well plates and incubated overnight. Before transfection, the cell culture medium was replaced with serum-free medium. DNA complexes, prepared as above (2 µg of DNA in each well), were added and incubated with cells for 4 h. Then, the transfection medium was replaced with FBS-containing medium and the cells were incubated for the next 48 h.
The β-galactosidase activity in cell extracts was measured with methyl-umbelliferyl-β-D-galactopyranoside (MUG) and normalized by the total protein concentration, measured with Bradford reagent (Helicon, Moscow, Russia), as described previously [30 (link)].
For the competition transfection study a 10-fold excess of cyclo(RGDfK) peptide was added to the cells 15 min before complex treatment, followed by the procedures described above.
GFP expression was determined by flow cytometry using a BD FACS-Canto II cytofluorimeter. Transfection efficacy was evaluated as a percentage of GFP-positive cells.
The β-galactosidase activity in cell extracts was measured with methyl-umbelliferyl-β-D-galactopyranoside (MUG) and normalized by the total protein concentration, measured with Bradford reagent (Helicon, Moscow, Russia), as described previously [30 (link)].
For the competition transfection study a 10-fold excess of cyclo(RGDfK) peptide was added to the cells 15 min before complex treatment, followed by the procedures described above.
GFP expression was determined by flow cytometry using a BD FACS-Canto II cytofluorimeter. Transfection efficacy was evaluated as a percentage of GFP-positive cells.
4
Peptide-DNA Transfection and Cell Viability
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PANC-1, HeLa or 293T cells were seeded at a density of 6 × 104 cells/well in 48-well plates. Peptide/DNA complexes were prepared with YOYO-1 iodide (1 molecule of the dye per 50 base pairs). Transfection was performed as described above. After 2 h of the complexes’ treatment, the cells were washed twice in 1× PBS (pH 7.2) and once with 1M NaCl (in 1× PBS). Then the cells were detached, resuspended and incubated with propidium iodide solution (50 µg/mL in 1× PBS) for 15 min in the dark to exclude dead cells. Subsequently, the cells were processed by flow cytometry with a BD FACS-Canto II cytofluorimeter. The results were presented as RFU/cell. A total of 10,000 living cells were taken into account.
5
Plasmid Transfection and Flow Cytometry
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PANC-1 cells were seeded at a density of 6 × 104 cells/well in 48-well plates. Before formulation of the complexes, DNA was labeled with YOYO-1 iodide (Thermo Fisher Scientific, Waltham, MA, USA) (1 molecule of the dye per 50 base pairs). Transfection was performed as described in subchapter 2.10. After 2 h of the complexes treatment, the cells were washed twice in 1× PBS (pH 7.2) and once with 1 M NaCl (in 1× PBS). Then, the cells were detached, resuspended and incubated with propidium iodide solution (50 µg/mL in 1× PBS) for 15 min in the dark to exclude dead cells. Subsequently, the cells were processed by flow cytometry with a BD FACS-Canto II cytofluorimeter. The results were presented as RFU/cell. 10,000 living cells were taken into account.
6
Quantitative Transfection Efficiency Assay
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PANC-1 or 293T cells were seeded at a density of 5 × 104 cells/well in 48-well plates a day before the experiments. Peptide/DNA complexes were formed with added YOYO-1 iodide based on 1 molecule of the dye per 50 base pairs. Transfection was performed according to the protocol described above. After 2 h of incubation with complexes, the cells were washed three times in 1× PBS (pH 7.2) and once with 1M NaCl (in 1× PBS). The cells were detached and incubated with propidium iodide solution, as described previously [34 (link)]. Then, the living cells, at a rate of 10,000 per sample, were analyzed by flow cytometry with a BD FACS-Canto II cytofluorimeter. The results were presented as RFU/cell.
7
Apoptosis and Cell Cycle Analysis
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Apoptosis was measured via flow cytometry, with annexin V-647 and propidium iodide (PI) staining. Briefly, the cells were washed twice in wash buffer (0.01 M Hepes, 0.14 M NaCl, and 2.5 mM CaCl2) and stained with annexin-V-647 for 15 minutes at room temperature, followed by a final wash and the addition of 2 μg/μL of PI. Viability of cells was assessed by gating PI+ vs PI− cells.
Cell cycle was measured flow-cytometrically using PI. Briefly, cells were washed in PBS and fixed in 70% ethanol at 4°C overnight. The following day the cells were washed thrice with PBS and incubated with PI (1:2000). Data were acquired using FACSCanto II cytofluorimeter and processed with FACSDiva 8.0 software (BD Biosciences).
Cell cycle was measured flow-cytometrically using PI. Briefly, cells were washed in PBS and fixed in 70% ethanol at 4°C overnight. The following day the cells were washed thrice with PBS and incubated with PI (1:2000). Data were acquired using FACSCanto II cytofluorimeter and processed with FACSDiva 8.0 software (BD Biosciences).
8
Cytometric analysis of PD-1 expression
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FCM was performed by a standard method and the results were acquired using a FACSCanto II cytofluorimeter (BD Biosciences) and analyzed using BD CellQuest Pro software (BD Biosciences). The following antibodies were used to measure the expression of PD-1 in the PBMCs: Mouse monoclonal anti-human-CD3-peridinin (Percp) (Catalogue number: 347344, clone: SK7), monoclonal mouse anti-human-CD4-fluorescein isothiocyanate (FITC) (cat. no.: 340962, clone: SK3), monoclonal mouse anti-human-CD8-allophycocyanin-cyanine 7 (Cy7) (cat. no.: 557760, clone: RPA-T8) and monoclonal mouse anti-human-PD-1-phycoerythrin (PE)-Cy7 (cat. no.: 561272, clone: EH12.1). The mAb was used to measure the purity of CD8+ T and NK cells separated by MACS, including monoclonal mouse anti-human-CD8-Percp (cat. no.: 341004, clone: SK7), monoclonal mouse anti-human-CD3-PE-Cy7 (cat. no.: 557749, clone: SP34-2) and monoclonal mouse anti-human-CD56-FITC (cat. no.: 340410, clone: NCAM16.2). The mouse IgG-FITC isotype control antibodies were also used. The antibodies used in FCM were all purchased from BD Pharmingen (San Diego, CA, USA).
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