To analyze cell surface protein expression, cells (3 × 105) were first incubated in 100 μL of primary antibody solution diluted in PBS plus 3% BSA and 1 mM EDTA. After incubation at 4°C in the dark for 30 minutes, the cells were washed once with 1 mL cold PBS. If a secondary antibody was to be applied, the incubation procedure was repeated. The cells were finally resuspended in 0.5 mL PBS. The disruption of the cell membrane upon viral infection was detected by using 8 μM ethidium homodimer 1 (Sigma-Aldrich) in PBS for 15 minutes at room temperature before acquiring the cells events. The stained cells were then acquired using the flow cytometer FACS Calibur (BD Biosciences), Gallios (Beckman-Coulter), or BD Celesta (BD Biosciences). FlowJo V10 (FlowJo, LLC) was used for the analysis. Table 1 shows the antibodies and working conditions used.
Ethidium homodimer 1
Manufactured by Merck Group
Sourced in United States, Switzerland, United Kingdom
Ethidium homodimer-1 is a fluorescent dye used in molecular biology applications. It is a small molecule that binds to DNA and emits fluorescence upon excitation. The core function of ethidium homodimer-1 is to detect and visualize nucleic acids, such as DNA and RNA, in various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Calcein am, by Merck Group (9 mentions)
Calcein am, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Facscalibur, by BD (1 mentions)
Celesta, by BD (1 mentions)
Clsm 510, by Zeiss (1 mentions)
Lsm 510, by Zeiss (1 mentions)
Calcein, by Merck Group (1 mentions)
Mg132, by Merck Group (1 mentions)
Rabbit anti lc3b, by Cell Signaling Technology (1 mentions)
Lactacystin, by Merck Group (1 mentions)
Rabbit anti t bet h 210, by Santa Cruz Biotechnology (1 mentions)
Goat anti actin 1 19, by Santa Cruz Biotechnology (1 mentions)
Ifn γ, by ProSpec (1 mentions)
Eclipse ts100 inverted microscope, by Nikon (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Tcs lsi, by Leica (1 mentions)
Normal human dermal fibroblasts (nhdf), by Lonza (1 mentions)
Non essential amino acids (neaa), by Sartorius (1 mentions)
Penicillin streptomycin antibiotics, by Sartorius (1 mentions)
21 protocols using ethidium homodimer 1
1
Cell Surface Protein Expression Analysis
2
Biomaterial Cell Viability Evaluation
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To determine the morphology and viability of cells within the biomaterial live/dead (L/D), staining was performed after 7 and 28 days of culture. Therefore, HA and CS samples were stained with 10 μM calcein-AM and 5 μM ethidium homodimer-1 (both Sigma-Aldrich, Buchs, Switzerland). After 1-hour incubation, the samples were imaged using confocal laser scanning microscopy (CLSM 510; Carl Zeiss, Germany). To quantify the number of L/D cells, 3 images were taken from 3 different fields of view, and a minimum of 100 cells were counted using image J (Wayne Rasband, NIH, USA). To visualize the expression of F-actin, additional staining with phalloidin and nuclear staining (4′,6-diamidino-2-phenylindole [DAPI]) was performed followed by CLSM.
3
Cytocompatibility of HA-Tyr under H2O2 and HRP
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A cytocompatibility range of HA-Tyr under varying H2O2 (50–1200 µmol) and HRP (0.1–1 U/mL) concentrations was identified with monolayer chondrocytes using a CellTiter-Blue assay. After 24 h and 96 h of incubation in chondropermissive medium, a CellTiter-Blue assay was performed according to the manufacturer’s instructions. After 1 h incubation in an atmosphere of 5% CO2 and 37 °C, fluorescence was measured using a TECAN plate reader (Tecan, Männedorf, Switzerland).
To visualize the cell viability, live/dead-staining was performed after 1, 7 and 28 days of culture in chondropermissive medium. SM and SMHA were washed with PBS and stained with 10 μM calcein (Sigma-Aldrich, St. Louis, MO, USA) and 5 μM ethidium homodimer-1 (Sigma-Aldrich, Buchs, Switzerland). After 1 h of incubation at 37 °C in a humidified atmosphere of 5% CO2, samples were imaged using laser scanning microscopy (LSM 510, Carl Zeiss, Germany). To quantify the number of live and dead cells, three images were taken from three different fields of view and a minimum of 100 cells were counted using image J software (Wayne Rasband, NIH, USA).
To visualize the cell viability, live/dead-staining was performed after 1, 7 and 28 days of culture in chondropermissive medium. SM and SMHA were washed with PBS and stained with 10 μM calcein (Sigma-Aldrich, St. Louis, MO, USA) and 5 μM ethidium homodimer-1 (Sigma-Aldrich, Buchs, Switzerland). After 1 h of incubation at 37 °C in a humidified atmosphere of 5% CO2, samples were imaged using laser scanning microscopy (LSM 510, Carl Zeiss, Germany). To quantify the number of live and dead cells, three images were taken from three different fields of view and a minimum of 100 cells were counted using image J software (Wayne Rasband, NIH, USA).
4
Evaluating Cell Viability in Scaffolds
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To assess cell viability, scaffolds were loaded with calcein acetoxymethyl ester (calcein AM; 1 μmol/L) and ethidium homodimer-1 (4 μmol/L) (Sigma-Aldrich) for 50 min at 37°C, on a 3D XYZ shaker. Scaffolds were then washed thrice with PBS and visualized using a confocal microscope. Cell viability was assessed before implantation.
5
Immunophenotyping and Autophagy Analysis
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IFNγ was purchased from ProSpec (East Brunswick, NJ). Ethidium homodimer 1, MG132 and Lactacystin were obtained from Sigma-Aldrich (St. Louis, MO). Brefeldin A was purchased from eBiosceince (San Diego, CA). Antibodies used in the studies were as follows: goat anti-actin (I-19) and rabbit anti-T-bet(H-210) from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit anti-LC3B (where LC3B is an isoform of the autophagy marker protein light chain 3) from Cell Signaling Technology (Danvers, MA); rat anti-Mouse CD45 APC-eFluor 780, rat anti-Mouse CD4 eFluor 450, rat Anti-Mouse CD8a PerCP-Cyanine5.5, rat Anti-Mouse CD11b APC, American hamster Anti-Mouse CD11c Alexa Fluor 488, American hamster anti-Mouse CD3e FITC, mouse anti-Mouse NK1.1 PE-Cyanine7, rat Anti-Mouse Ly-6G (Gr-1) eFluor 450, mouse anti-mouse MHC I (H-2Kd) APC and mouse IgG2a APC from eBioscience; Rabbit anti-CD3 [SP7] antibody from abcam (Cambridge, MA).
6
Fibroblast Encapsulation in DDA-Gelatin Hydrogels
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Example 6
Normal human dermal fibroblasts (NHDF) (Lonza, Basel, Switzerland) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, Paisley, UK) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Cramlington, UK), 1% penicillin/streptomycin antibiotics (Biological Industries, Beit-Haemek, Israel), 1% non-essential amino acid solution (NEAA) (Biological Industries), and 0.2% ME (Sigma-Aldrich). Sterilization of DDA precursor solutions was done using a 0.22 μm syringe filter, whereas gelatin precursor solutions were UV-treated for 20 min. Cells were suspended in DDA and then mixed with gelatin to form 0.5 ml hydrogel samples. The final seeding density was 1×106 cells/ml. Cell-embedded hydrogels were then incubated for 1 hour at 37° C. to allow for chemical crosslinking before addition of media. Culture media were replaced three times during the one-week culture period. Images were taken during the culture period using an Eclipse TS100 inverted microscope (Nikon Instruments, Melville, N.Y., USA). Viability assays were conducted using calcein AM (cytoplasmic marker) and ethidium homodimer-1 (nucleic acids dye) (Sigma-Aldrich), according to standard protocols. Images of cells within the whole constructs were taken using a super-zoom macro-confocal microscope (Leica TCS LSI, Wetzlar, Germany).
7
Cell Viability of 3D Printed Constructs
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The printed constructs were assessed for cell viability and cell distribution on days 0, 1, 4, 7 and 14 subsequent to printing. Printed constructs were washed in HBSS before suspending in 0.5 mL HBSS containing 1 µM Calcein-AM (eBioscience brand, ThermoFisher Scientific, Loughborough, UK) and 2 µM Ethidium Homodimer 1 (Sigma-Aldrich, Gillingham, UK). Constructs were incubated for 30 min at 37 °C before washing twice with HBSS and capturing fluorescent images using a Leica DM IL LED microscope (Leica, UK). Cell viability was calculated from images using ImageJ (1.48v) software (Schneider et al., 2012). Gross images were also captured on each of these days to establish printed construct stability.
8
Live-Dead Staining Cell Viability
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Viable cell number was assessed following gel dissolution by live-dead staining. Cells were stained with 1 µM Calcein-AM (eBioscience brand, ThermoFisher Scientific, Loughborough, UK) and 2 µM Ethidium Homodimer 1 (Sigma-Aldrich, Gillingham, UK) for 15 min at 37 °C. Viable cell number and percentage viability were assessed using a Countess II FL automated cell counter (Invitrogen brand, ThermoFisher Scientific).
9
Cell Viability Quantification in Hydrogels
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Cell viability was visualized and quantified as previously described [34] (link). At days 1 and 21, cell-seeded hydrogels were washed 3X in phosphate buffered saline (PBS; Life Technologies) for 5 mins and then incubated in 4 µM calcein-AM (EMD Millipore, Billerica, MA) and 4 µM ethidium homodimer-1 (Sigma) in PBS for 45 mins. Constructs were washed 3X in PBS prior to imaging on a Nikon A1 Confocal Microscope (Nikon Instruments, Melville, NY). Cell viability was quantified using ImageJ software (National Institute of Health, Bethesda, MD).
10
β-TCP Scaffold for Cell Culturing
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β-TCP powder was purchased from Kunshan Chinese Technology New Materials Corporation (Kunshan, China). Cetyltrimethylammonium bromide (CTAB), ammonium fluoride (NH4F), tetraethoxysilane (TEOS), copper nitrate trihydrate, pluronic F-127, calcein AM, ethidium homodimer-1, fluorescein isothiocyanate (FITC), and 4,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (Shanghai, China). Cell Counting Kit-8 (CCK-8) was purchased from Beyotime Biotechnology (China).
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