Cells adhered on slides previously treated with polylysine were fixed with 4% paraformaldehyde for 25 min and then permeabilized with 0.1% Triton for 10 min. Cells were washed three times with PBS and incubated with anti-GAPDH diluted 1:800 in PBS-0.1% BSA for 1 h. After three washes with PBS, cells were incubated with anti-mouse antibody conjugated to Alexa 555 diluted 1:500 and 5μg/ml of DAPI in PBS-0.1% BSA for 45 min. In the case of FISH coupled assay, cells were then post-fixed with 4% cold formaldehyde for 20 min and washed three times with PBS. Cells were dehydrated with 70%, 80% and 90% ethanol and then air-dried. 10μl of hybridization solution (70% formamyde, 20 mM Tris-HCl pH 7.0 and 1% BSA) containing 1nmol of telomeric probe (5`TTAGGGTTA3’ FITC PNA) was added and slides were sealed using Frame-Seal (Bio-Rad). Slides were maintained at 95°C for 5 min and then incubated at 37°C overnight and then washed with Wash solution from Telomere PNA kit/FITC (Dako) for 5 min. Slides were again treated with 70%, 80% and 90% ethanol and air-dried. Slides were sealed in the presence of Vecta shield (Vector). Images were acquired through z-series of 0.2μm using lens of 100X 1.35NA using Cell R software in Olympus IX81 microscopy. Images were deconcolved using Autoquant X2.1.
Telomere pna kit fitc
Manufactured by Agilent Technologies
Sourced in Denmark
The Telomere PNA kit/FITC is a laboratory equipment product designed for the measurement of telomere length. It utilizes a peptide nucleic acid (PNA) probe conjugated with a fluorescein isothiocyanate (FITC) dye to enable the visualization and quantification of telomeric DNA sequences.
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Lab products found in correlation
Vectashield, by Vector Laboratories (1 mentions)
Frame seal, by Bio-Rad (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Tb2 thermoblock, by Analytik Jena (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Fcs express version 3, by De Novo Software (1 mentions)
Anti cd25 microbeads, by Miltenyi Biotec (1 mentions)
Anti cd45ra, by Miltenyi Biotec (1 mentions)
Macsquant, by Miltenyi Biotec (1 mentions)
Telotaggg telomere length assay kit, by Roche (1 mentions)
Coulter epics xl, by Beckman Coulter (1 mentions)
13 protocols using telomere pna kit fitc
1
Immunofluorescence and FISH Assay Protocol
2
Telomere Length Measurement by Flow Cytometry
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Telomere length was measured by telomere PNA kit/FITC (DAKO)24 (link). In brief, the sample DNA was denatured for 10 minutes at 82 °C in a microcentrifuge tube either in the presence of hybridization solution without probe or in hybridization solution containing fluorescein‐conjugated PNA telomere probe. Then hybridization took place in the dark at room temperature (RT) overnight. The hybridization was followed by two 10‐minute post‐hybridization washes with a Wash Solution at 40 °C. The sample was then resuspended in an appropriate buffer for further flow cytometric analysis. DNA Staining Solution included in the kit was used for identification of G0/G1 cells. After flow cytometric analysis, the data obtained were used for determination of a relative telomere length (RTL). The RTL value was calculated as the ratio between the telomere signal of each sample and the control cells with correction for the DNA index of G0/G1 cells.
3
Telomere Length Measurement by Flow Cytometry
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Telomere length was measured by means of flow cytometry with the use of a commercial Telomere PNA Kit/FITC (Dako) according to the user manual. Detection of the samples labeled with fluorescein-conjugated peptide nucleic acid was done with the use of the MACSQuant Analyzer X. As recommended, the cell line 1301 was used as internal control and relative telomere length (RTL) was calculated as follows:
RTL = (mean FL1 sample cells with probe – mean FL1 sample cells without probe) × 2 × 100/(mean FL1 control cells FL1 control cells with probe–mean without probe).
RTL = (mean FL1 sample cells with probe – mean FL1 sample cells without probe) × 2 × 100/(mean FL1 control cells FL1 control cells with probe–mean without probe).
4
Measuring Telomere Length in Cell Lines
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Telomere length was measured using Telomere PNA Kit/FITC (DAKO, Glostrup, Denmark; catalogue #K5327). Telomere length of the HPDE-1/E6E7 cells expressing either Mock or (P)RR was determined. Human T-cell leukaemia 1301 cells (ECACC, Salisbury, UK; catalogue #EC01051619-G0) characterised by tetraploidy and long telomeres were used as an internal standard. Test and control cells were mixed at a ratio of 1:1 and a total of 4 × 106 cells was resuspended in 300 µL of hybridization solution with or without a fluorescein isothiocyanate (FITC)-conjugated telomere probe. DNA was denatured for 10 min at 82 °C. In situ hybridization was performed at room temperature under dark conditions. Cells were rinsed with washing solution and heated for 10 min at 40 °C. They were then resuspended in 0.5 mL of DNA staining solution containing propidium iodide and RNase A and incubated for 3 h at 4 °C in the dark. Samples were analysed by flow cytometry. FlowJo (Tomy Digital Biology Co., Ltd., Tokyo, Japan) was used for analysis and relative telomere length (RTL) was calculated based on mean fluorescence intensity (MFI)72 (link).
5
Measuring Telomere Length by Flow Cytometry
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Telomere length was measured with the telomere PNA kit/FITC (DAKO Denmark, Glostrup, DK). In brief, the sample DNA was denatured for 10 min at 82 °C either in the presence of hybridization solution without probe or in hybridization solution containing a fluorescein-conjugated PNA telomere probe. The hybridization then took place in the dark at room temperature overnight, followed by two 10-min post-hybridization washes with a wash solution at 40 °C. The sample was then resuspended in the appropriate buffer for further analysis by flow cytometry. The DNA Staining Solution included in the kit was used for the identification of G0/1 cells. After flow cytometry analysis, the data obtained were used to determine the relative telomere length (RTL). The RTL value was calculated as the ratio between the telomere signal of each sample and the control cells (1301 cell line, European Collection of Cell Cultures) with correction for the DNA index of G0/1 according to manufacturer’s instructions.
6
Telomere Length Quantification by Flow Cytometry
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The analysis was conducted according to the telomere PNA kit/FITC used for flow cytometry (Dako, Carpinteria, CA, USA). A total of 1 and/or 2×106 sorted cells and control cells were diluted in PBS and divided into two tubes, namely A and B. The DNA was denatured at 82°C for 10 min in an Eppendorf tube in the presence of hybridization solution with or without a fluorescein-conjugated PNA telomere probe. Subsequently, hybridization was performed in the dark at room temperature overnight. The samples were washed twice in washing solution at 40°C for 10 min each. Finally, the cells were resuspended in DNA-staining solution and stored in the dark at 2–8°C for 2–3 h prior to flow cytometric analysis. The specific fluorescence activity was proportional to the telomere staining and was detected in FL1, whereas the fluorescence derived from DNA staining was detected in FL3. Finally, at least 20,000 cells were acquired and analysed using the fluorescence-activated cell sorter (FACSCalibur) flow cytometer (BD Biosciences). The DNA index of the cells was determined as follows: RTL = (mean FL1 sample cells with probe-mean FL1 sample cells without probe) × DNA index of control cells × 100/(mean FL1 control cells with probe-mean FL1 control cells without probe) × DNA index of sample cells.
7
Telomere Length Quantification by Flow Cytometry
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Telomere length was measured by telomere PNA kit/FITC (DAKO). In brief, the sample DNA was denatured for 10 minutes at 82°C in a microcentrifuge tube either in the presence of hybridization solution without probe or in hybridization solution containing fluorescein‐conjugated PNA telomere probe. Then hybridization took place in the dark at room temperature (RT) overnight. The hybridization was followed by two 10‐minute post‐hybridization washes with a Wash Solution at 40°C. The sample was then resuspended in an appropriate buffer for further flow cytometric analysis. DNA Staining Solution included in the kit was used for identification of G0/1 cells. After flow cytometric analysis, the data obtained were used for determination of a relative telomere length (RTL). The RTL value was calculated as the ratio between the telomere signal of each sample and the control cell (1301 cell line) with correction for the DNA index of G0/1 cells.
8
Telomere Length Quantification in T Cells
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Telomere PNA kit/FITC (Dako, São Paulo, Brazil) was used following the manufacturer’s instructions, including thymocytes from 6-week old mice as reference for normalization. Briefly, samples were prepared by mixing 106 mouse thymocytes and 106 TCM cells. The mixture was distributed into four tubes. 150 μl of FITC-labeled peptide nucleic acid (PNA) probe solution was added into two tubes while 150 μl of unlabeled PNA probe solution was added into the other two. Samples were hybridized in a pre-warmed heating block (TB2 Thermoblock, Biometra, Göttingen, Germany) set at 82 °C, 10 min, and left overnight at room temperature. Samples were washed twice. Between washing steps, samples were heated to 40 °C in a pre-warmed TB2 Thermoblock (Biometra) for 10 min. Samples were resuspended in 250 μL of DNA staining solution (1X), and stored overnight at 4 °C, away of light. Then, samples were analyzed by flow cytometry in a FACSCanto II (BD Biosciences).
9
Comprehensive T Cell Phenotyping and Analysis
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Human T cells were analyzed by flow cytometry after staining with fluorochrome-conjugated monoclonal antibodies (mAbs) to CD8 (SKI), CD62L (Dreg-56), CD45RO (UCHL1), CD45RA (L48), CD28 (CD28.2), CD3 (SK7), IFN-γ (XMG1.2), CD107a (H4A3), KLRG1 (2 F1/KLRG1), CD57 (HNK-1), Tim3 (F38-2E2), PD1 (EH12.1), LAG3 (FAB2319C), and phosphorylated Akt (pAkt, pS473)(M89-61) (BD Biosciences). Data acquisition was performed on a MACSQuant (Miltenyi Biotec) and analyzed using FCS Express, Version 3 software (De Novo Software). Telomere length analysis was performed using the flow-FISH technique with Telomere PNA Kit/FITC obtained from Dako (Dako Denmark A/S, Denmark). Biotinylated Erbitux (cetuximab) and streptavidin-PE were used to identify T cells that expressed truncated human EGFR (EGFRt). Anti-CD4 microbeads were purchased from Stemcell Technologies, and anti-CD14, anti-CD45RA, and anti-CD25 microbeads were purchased from Miltenyi Biotec. Proliferation capacity was determined by labeling the cells with carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen).
10
Telomere Length Measurement by Flow Cytometry
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