The cellular uptake of DOX-loaded PLA–PEG–FA SPIONs was evaluated in HeLa and CT26 cells using flow cytometry and fluorescence microscopy. For the flow cytometry, the cells were seeded in 12-well plates in 1 mL of growth medium at the initial density of 5 × 104 cells per well and cultured for 24 hrs. The medium was then changed with a 1 mL fresh suspension containing different samples. Then, the cells were collected and washed three times with PBS for analyzing the DOX fluorescence intensity using a FACS caliber flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA). Moreover, cellular internalization of the DOX-loaded PLA–PEG–FA SPIONs and free DOX were analyzed by a fluorescence microscope (Leica Microsystems Inc., Buffalo Grove, IL). To identify necrosis and apoptosis, the Annexin V-FITC apoptosis detection kit was used. Briefly, the cells were incubated with the different samples for 24 hrs. Then, the cells were harvested, centrifuged and washed with PBS. Afterwards, the cells were re-suspended in 100 μL of binding buffer containing 5 μL FITC-labelled annexin V and incubated for 20 min at ambient temperature in a dark room. Finally, the cells were examined using a FACS caliber flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA).
Facs caliber flow cytometer
Manufactured by BD
Sourced in United States, Germany, China, United Kingdom
The BD FACS Caliber flow cytometer is a laboratory instrument designed to analyze and sort cells and particles in a fluid suspension. It uses laser technology to detect and measure physical and fluorescent characteristics of cells or particles as they pass through a flow cell. The core function of the FACS Caliber is to provide quantitative data on the size, granularity, and fluorescence intensity of individual cells or particles within a sample.
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Lab products found in correlation
Cellquest software, by BD (40 mentions)
Cellquest pro software, by BD (9 mentions)
Cell cycle detection kit, by Keygen Biotech (8 mentions)
Propidium iodide, by Merck Group (7 mentions)
Modfit software, by Verity Software House (6 mentions)
Cellquest pro, by BD (5 mentions)
Cellquest, by BD (5 mentions)
Propidium iodide, by BD (4 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (4 mentions)
Modfit software, by BD (4 mentions)
Cycletest plus dna reagent kit, by BD (4 mentions)
Annexin 5, by BD (3 mentions)
Apo brdu tunel assay kit, by Thermo Fisher Scientific (3 mentions)
Trypsin, by Thermo Fisher Scientific (3 mentions)
Cell based ros assay kit, by Beyotime (3 mentions)
Annexin 5 fitc, by BD (3 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (3 mentions)
Annexin 5 pe 7aad apoptosis kit, by BD (2 mentions)
Alexa488 alexa594 conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Lsr 2, by BD (2 mentions)
317 protocols using facs caliber flow cytometer
1
DOX-loaded PLA-PEG-FA SPION Uptake and Cytotoxicity
2
Analysis of CD206 Expression
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At 48 hrs post-treatment, cells were scratched from 6-well plates, centrifuged and resuspended. Non-specific labeling was blocked with anti-CD16/32 (Fc Block) (BioLegend, San Diego, CA, USA) before specific labeling. For analysis of CD206 expression, cells were fixed, permeabilized, and incubated with PE anti-mouse CD206 (MMR) (BioLegend, San Diego, CA, USA) following the manufacturer’s protocol. In all cases, appropriate antibody iso-type controls were used. Flow cytometry was performed on a BD FACS Caliber flow cytometer (BD Biosciences, San Jose, CA, USA). FlowJo software was used for acquiring and analyzing the data. Experiments from each group were repeated at least 3 times, and representative data from each group are shown.
3
Characterization of Mesenchymal Stem Cells
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After second consecutive passages, to measure the
cell surface markers of MSCs and to confirm their
development, we used the following conjugated
antibodies: PE Mouse Anti-Rat CD44, PE Mouse Anti-
Rat CD73, PE Mouse Anti-Rat CD105, FITC Mouse
Anti-Rat CD90, PE Mouse Anti-Rat CD34, PE Mouse
Anti-Rat CD45, PE Mouse Anti-Rat CD11b, and isotype
control antibody. After trypsinization of the cells with
0.25% trypsin/ ethylenediaminetetraacetic acid (EDTA)
solution, they were re-suspended in PBS and counted
using hemocytometer. A number of 1×106 cells were
incubated in fluorochrome-conjugated antibody at a
dilution ratio of 1:10 at room temperature for 20 minutes
in the dark place. The stained ADSCs were analyzed using
a BD FACSCaliber™ flow cytometer (BD Biosciences,
USA) and the FlowJo software (version 10.4). A total of
200,000 cells were assessed in each sample.
cell surface markers of MSCs and to confirm their
development, we used the following conjugated
antibodies: PE Mouse Anti-Rat CD44, PE Mouse Anti-
Rat CD73, PE Mouse Anti-Rat CD105, FITC Mouse
Anti-Rat CD90, PE Mouse Anti-Rat CD34, PE Mouse
Anti-Rat CD45, PE Mouse Anti-Rat CD11b, and isotype
control antibody. After trypsinization of the cells with
0.25% trypsin/ ethylenediaminetetraacetic acid (EDTA)
solution, they were re-suspended in PBS and counted
using hemocytometer. A number of 1×106 cells were
incubated in fluorochrome-conjugated antibody at a
dilution ratio of 1:10 at room temperature for 20 minutes
in the dark place. The stained ADSCs were analyzed using
a BD FACSCaliber™ flow cytometer (BD Biosciences,
USA) and the FlowJo software (version 10.4). A total of
200,000 cells were assessed in each sample.
4
Cellular Uptake of PAMAM-NH2-FITC
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The MCF-7 and MCF-7/ADR cells were preincubated with brefeldin A, monensin, and bafilomycin A1 at a given concentration18 (link) (Table 1 ) for 30 minutes in a humidified atmosphere with 5% CO2 at 37°C, and incubated with PAMAM-NH2-FITC (10 μg/mL) for another 3 hours. The cells were then processed and examined using a BD FACS Caliber flow cytometer.
5
Cellular Uptake of PAMAM-NH2-FITC
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The MCF-7 cells and MCF-7/ADR cells were seeded separately in six-well culture plates (1×106 cells/well) in a humidified atmosphere with 5% CO2 at 37°C. After 24 hours, the cells were preincubated at 4°C for 1 hour or with sodium azide and 2-deoxyglucose for 1 hour at 37°C. Subsequently, PAMAM-NH2-FITC (10 μg/mL) was added and incubated at 4°C or 37°C for an additional 3 hours. Finally, the samples were disposed and analyzed using a BD FACS Caliber flow cytometer.
6
Cellular Internalization Assay of PAMAM-FITC
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The MCF-7 and MCF-7/ADR cells were seeded separately in six-well culture plates in a humidified atmosphere with 5% CO2 at 37°C for 24 hours. The cells were then incubated with PAMAM-NH2-FITC (10 μg/mL) in a fresh serum-free medium for 3 hours, rinsed three times with cold PBS, and incubated in a fresh medium containing various inhibitors18 (link) (the intracellular transport inhibitors in Table 1 ) for another 5 hours. Finally, the cells were washed with PBS and resuspended in PBS, and the samples were analyzed with a BD FACS Caliber flow cytometer.
7
Cellular Uptake Dynamics of PAMAM Dendrimers
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The MCF-7 and MCF-7/ADR cells were seeded separately in six-well culture plates at a density of 1×106 cells/well in a humidified atmosphere with 5% CO2 for 24 hours at 37°C and incubated with PAMAM-NH2-FITC (10 μg/mL) for 3 hours. The cells were rinsed with PBS and immediately incubated with a fresh dendrimer-free medium for different lengths of time. Finally, the cells were washed with PBS and resuspended in PBS, and the samples were analyzed with a BD FACS Caliber flow cytometer.
8
RA-FLSs Apoptosis Assessment
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RA-FLSs (1.5 × 105 / well) treated as indicated were collected and stained with Annexin V-FITC/propidium iodide (PI) Apoptosis Detection kit (Elabscience, China). Cell death analysis was performed on the BD FACS Caliber flow cytometer.
9
Cell Proliferation and Apoptosis Assay
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Cells were seed in 96 well plates and incubated in DMSO (control) or different concentration of CX-5461 for 3 days. CellTiter 96 AQueous One Solution Cell Proliferation solution (Promega) was added to each well and incubated for 1 h at 37°C in dark. Absorbance was recorded at 490 nm using Bio-Rad microplate reader. Results were background subtracted and normalized to DMSO treated control. Experiment was repeated three times and results were plotted as mean +/− S.D.
Annexin V was used for measuring apoptosis (BD Biosciences). Patient samples or cell lines were seeded in 6 well plates and incubated with DMSO or CX-5461. After 48 to 72 h cells were harvested, washed in PBS and suspended in Annexin V binding buffer. Annexin V was added to each sample and incubated in dark for 30 min. Cells were analyzed on BD FACScaliber flow cytometer. Results were normalized to control and plotted as mean +/− S.D. of three separate experiments.
Annexin V was used for measuring apoptosis (BD Biosciences). Patient samples or cell lines were seeded in 6 well plates and incubated with DMSO or CX-5461. After 48 to 72 h cells were harvested, washed in PBS and suspended in Annexin V binding buffer. Annexin V was added to each sample and incubated in dark for 30 min. Cells were analyzed on BD FACScaliber flow cytometer. Results were normalized to control and plotted as mean +/− S.D. of three separate experiments.
10
Quantifying STRO-1+CD117+ Cells in Osteosarcoma
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Flow cytometry was performed to examine the proportions of STRO-1+CD117+ cells in U2OS and 143B cells as previous described [18 (link)]. U2OS and 143B cells were cultured in DMEM medium, and the cell suspension (106 cells/100 μL) was incubated with 10 μL STRO-1-PE or CD117-APC (Abcam) at 4°C for 30 min in darkness. After that, the proportions of STRO-1+CD117+ cells in U2OS and 143B cells were detected using a BD FACScaliber flow cytometer (BD Biosciences, San Jose, CA, USA). The fluorescent intensity was analyzed using Cell Quest Software (BD Biosciences).
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