Senescent and nonsenescent HDFs were seeded at 35 × 105 cells in 100-mm dishes. After an overnight incubation, the cells were treated with nintedanib (5 μM) or ABT263 (5 μM) for 3 days. The cells were harvested and washed three times with PBS. Then, the harvested cells were resuspended in 100 μl of 1× Annexin V binding buffer (0.1 M HEPES (pH 7.4), 1.4 M NaCl, and 25 mM CaCl2) containing 5 μl of Annexin V-Alexa Fluor®350 conjugate (Thermo Fisher Scientific, A23202) and 5 μl of 100 μg/ml propidium iodide (PI). After 30 min of incubation in the dark, 400 μl of 1× Annexin V binding buffer was added (final volume: 500 μl) to each sample. The cells were analyzed using a flow cytometer (LSR Fortessa, BD) within 1 hr to determine the intensity of Annexin V staining (excitation wavelength of 488 nm with a 530/30 nm bandpass filter, DAPI channel) and that of PI (excitation wavelength of 561 nm with a 583/22 nm bandpass filter, PE channel). The results were analyzed using FlowJo (version 10).
Annexin 5 alexa fluor 350 conjugate
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Annexin V Alexa Fluor 350 Conjugate is a fluorescently labeled protein used to detect and quantify apoptosis in cells. It binds to phosphatidylserine, which is exposed on the cell surface during apoptosis. The Alexa Fluor 350 dye provides a blue fluorescent signal that can be detected using flow cytometry or fluorescence microscopy.
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Lab products found in correlation
Annexin binding buffer, by Thermo Fisher Scientific (3 mentions)
Propidium iodide, by Thermo Fisher Scientific (2 mentions)
Biotin conjugated anti ter119 antibody and magnetic beads, by STEMCELL (1 mentions)
Lsrii cytometer, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Uvm 340, by Harvard Bioscience (1 mentions)
Mtt solution, by Merck Group (1 mentions)
Lsr fortessa cytometer, by BD (1 mentions)
Cycletest plus dna kit, by BD (1 mentions)
5 protocols using annexin 5 alexa fluor 350 conjugate
1
Senescent Cell Apoptosis Assay
2
Annexin V and Propidium Iodide Apoptosis Assay
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Drug treated cells were harvested by trypsinization and resuspended in 1X Annexin V binding buffer (10 × Annexin V binding buffer: 0.1 M HEPES (pH 7.4), 1.4 M NaCl, 25 mM CaCl2) containing 5 μl of Annexin V-Alexa fluor®350 conjugate (Thermo Fisher Scientific, A23202) and 2 μl of 100 μg/ml propidium iodide per 1x105 cells in 100 μl, and then incubation for 15 min at the dark. After the incubation, add 400 μl 1X Annexin-binding buffer (final vol. 500 μl). The cells were analyzed by flow cytometry within 1 h.
3
Apoptosis and Proliferation Assessment in Spleen
4
Metabolic Activity and Viability of Dermal Fibroblasts
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DFs isolated from young LFD animals were cultured in monolayer condition or in coculture with keratinocytes nontransduced or transduced with Ad-Foxn1 or Ad-GFP (n = 5 per condition).
DF cell metabolic activity was measured using MTT colorimetric method as described previously [43 (link)]. Briefly, after 24 h and 48 h, sterile MTT solution (3-[4,5-dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium bromide; Sigma-Aldrich, St. Louis, MO, USA) was added to each well and incubated for 4 h. Next, the medium was removed and formazan crystals were dissolved in DMSO for 1 h. Absorbance was measured at 570 nm using a microplate reader (ASYS Hitech GmbH, UVM340, Biogenet; Biochrom Ltd., Cambridge, UK) and MicroWin 2000 software version V4.0 for Windows XP (Siemens, Monachium, Germany).
For the viability assay after 24 h of culture, cells were trypsinized, counted using the trypan blue exclusion method and resuspended in Annexin Binding Buffer (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) with addition of Annexin V Alexa Fluor 350 Conjugate (Life Technologies) and propidium iodide (Life Technologies). Samples were analyzed using flow cytometry with BD LSRFortessa and BD FACSDiva TM v6.2 software (Becton Dickinson, Franklin Lakes, NJ, USA).
DF cell metabolic activity was measured using MTT colorimetric method as described previously [43 (link)]. Briefly, after 24 h and 48 h, sterile MTT solution (3-[4,5-dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium bromide; Sigma-Aldrich, St. Louis, MO, USA) was added to each well and incubated for 4 h. Next, the medium was removed and formazan crystals were dissolved in DMSO for 1 h. Absorbance was measured at 570 nm using a microplate reader (ASYS Hitech GmbH, UVM340, Biogenet; Biochrom Ltd., Cambridge, UK) and MicroWin 2000 software version V4.0 for Windows XP (Siemens, Monachium, Germany).
For the viability assay after 24 h of culture, cells were trypsinized, counted using the trypan blue exclusion method and resuspended in Annexin Binding Buffer (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) with addition of Annexin V Alexa Fluor 350 Conjugate (Life Technologies) and propidium iodide (Life Technologies). Samples were analyzed using flow cytometry with BD LSRFortessa and BD FACSDiva TM v6.2 software (Becton Dickinson, Franklin Lakes, NJ, USA).
5
Keratinocyte Differentiation and Viability
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Non-transfected keratinocytes and keratinocytes transfected with Ad-Foxn1 or Ad-GFP were analysed for K10 and K14 expression using flow cytometry methods. Keratinocytes were trypsinized 48 hours after transfection, suspended in warm PBS and incubated for 30 min with the following antibodies: anti-keratin 10-PerCP (mouse monoclonal, clone SPM261; Novus Biologicals conjugated with PerCP; Lynx Rapid PerCP antibody conjugation kit; AbD Serotec) and anti-keratin 14-APC (mouse monoclonal, clone LL002; Novus Biologicals conjugated with APC; Lynx Rapid APC antibody conjugation kit; AbD Serotec). To determine the percentage of viable, apoptotic and necrotic cells, keratinocytes collected by trypsinization (0.05% trypsin-EDTA) were resuspended in Annexin Binding Buffer (Life Technologies). Then, Annexin V Alexa Fluor 350 Conjugate (Life Technologies) and propidium iodide (Life Technologies) were added. For characterization of the cell cycle phase distribution, a BD Cycletest Plus DNA Kit (BD Biosciences) was used according to the manufacturer’s description. Samples (n = 4 for keratin analysis, n = 8 for cell viability and n = 7 for cell cycle phase) were analysed using flow cytometry with BD LSR Fortessa Cytometer running Diva software 6.2 (BD Biosciences).
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