Cell viability in the printed constructs was assessed by live/dead assay (R37601; Life Technologies, Carlsbad, NM, USA) according to manufacturer’s instructions. Briefly, after printing, and crosslinking, samples were washed three times with PBS, stained with live green (A) (Calcium-AM; 0.5 μL/mL) and dead red (B) (ethidium homodimer; 2 μL/mL), and incubated for 15 min at RT. Fluorescence images of printed samples were captured 1 h and 24 h after deposition under confocal microscope (Olympus FV1200, Olympus, Tokyo, Japan). Data are representative of the printed samples of four layers stacked images.
R37601
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The R37601 is a laboratory centrifuge designed for general-purpose applications. It features a swing-out rotor that can accommodate a variety of sample tubes and microtiter plates. The centrifuge operates at a maximum speed of 4,000 rpm and provides precise speed and time controls.
Automatically generated - may contain errors
Lab products found in correlation
Fv1200, by Olympus (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Dm irb, by Leica (1 mentions)
Phosphate buffered saline (pbs), by Lonza (1 mentions)
Live dead kit, by Thermo Fisher Scientific (1 mentions)
Axio observer, by Zeiss (1 mentions)
Live dead cell dye, by Thermo Fisher Scientific (1 mentions)
35 mm confocal dish, by SPL Life Sciences (1 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
Matlab, by MathWorks (1 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
Epoch 2, by Agilent Technologies (1 mentions)
15 protocols using r37601
1
Cell Viability Assessment in Printed Constructs
2
MSC Viability on Biomaterial Substrates
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MSCs were seeded at 3.5 × 104 cells per well onto SMO, AE, SLA50, and SLA250 substrates in a 24‐well plate (n = 3). After 24‐h incubation at standard culture conditions, MSCs were washed twice in 1 mL of Dulbecco's phosphate buffered saline (PBS, Lonza, 17‐512F) and incubated with Live/Dead assay reagent for 15 min in the dark at RT (Life technologies, R37601). Following this, substrates were viewed using a fluorescence microscope fitted with the appropriate filters (Leica, DMIRB).
3
Live/Dead Assay of 3D-Printed Constructs
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Cell viability in the printed constructs was assessed by live/dead assay (R37601; Life Technologies, Darmstadt, Germany) according to manufacturer’s instructions. Briefly, live green (A) (Calcium-AM; 0.5 μL/mL) and dead red (B) (ethidium homodimer; 2 μL/mL) were prepared in culture media. Cross-linked samples were incubated for 15 min at RT. Fluorescence images of printed samples were captured 1 and 24 h after deposition under a laser scanning confocal microscope (Olympus FV1200, Olympus, Tokyo, Japan). Three independent samples were utilized for the assay (n = 3), with seven stacked images per sample (10 layers).
4
Viability Assay of Cultured Neurons
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Viability was analyzed after 3 DIV using a calcein and propidium iodide based live/dead kit per manufacturer's instructions (Life Technologies, R37601). For analysis, the first five non-overlapping fields of view per well, moving from the left well edge, were imaged at 20 × using standard epi-fluorescence fluorescein and rhodamine filter sets (Zeiss, Axio Observer). Experimentally blinded individuals analyzed live and dead cells using ImageJ (National Institutes of Health, Bethesda, MD, USA). Data represent triplicates from four experimental repeats, totaling at least 27 fields of view and at least 300 neurons per group as previously described (Faust et al., 2017 (link)). Significance between groups was determined by one-way analysis of variance (ANOVA) as noted in Section 2.13 .
5
Viability Quantification of Encapsulated Islets
6
Islet Viability Assessment Through Live/Dead Staining
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Fifty islets without MSCs, with MSC spheroids, or with eMSC spheroids were cultured in 3 ml of RPMI 1640 complete media for 24 hours in nonadherent 25-mm2 culture dishes. After culture, islets were handpicked and stained by calcein-AM (green, live) and ethidium homodimer (red, dead) according to the manufacturer’s protocol (R37601, Thermo Fisher Scientific). Fluorescent microscopic images were taken using a digital inverted microscope (EVOS FL Cell Imaging System). Quantification of the percentage of live cells in islets was carried out by calculating the intensity of fluorescence using ImageJ.
7
Quantifying SARS-CoV-2 Cytopathic Effects
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To determine CPE, cells were infected with HCoV-229E at the indicated MOIs and timepoints. After infection, cells were washed once with 1x PBS, fixed with 4% PFA for 10 minutes at room temperature, washed 3x with 1x PBS, stained with 0.1% Crystal Violet in dH2O for 10 minutes at room temperature, then rinsed repeatedly with dH2O. Cells were then solubilized in methanol for 10 minutes, then quantified using a plate reader reading absorbance at OD 570nm. To perform live/dead fluorescent microscopy to evaluate CPE, cells were infected at the indicated MOIs and timepoints, stained with a live/dead cell imaging kit (Thermo, R37601), and imaged immediately on a ZOE fluorescent microscope.
8
Live/Dead Cell Staining Assay
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All cell culture steps refer to Materials and methods 2.18, and only the seeding density was modified to 1 × 105 cells/well. Briefly, the cell culture plate obtained above were washed with PBS three times, and then the live/dead cell dye (R37601, Thermo Fisher Scientific, Germany) was added to the culture plate and incubated in a 37 °C humidified environment with 5% CO2 for 30 min. After staining, the cell culture plates were washed with PBS three times, and then the stained cells were observed by a fluorescence microscope, where the living cells showed green while the dead cells showed red. The statistical analysis was performed through ImageJ software.
9
Photodynamic Therapy of U87MG Cells
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To visualize the frequency of live cells after treatment with KUP-1 (0.4 mg mL−1) and O 2 @ KUP-1 (0.4 mg mL−1) under irradiation at 530 nm (60 mW cm-2) for 3 min, U87MG cells (2 × 104) were plated on a 35-mm confocal dish (SPL, Korea). When the cells were plated on the dish, they were treated with KUP-1 and O 2 @ KUP-1 in SF media. After incubation for 12 h, the cells were rinsed twice with 1 × PBS and irradiated with SF media at 530 nm. The cells on SF media were irradiated at 530 nm (60 mW cm−2) for 3 min, and the cells were incubated for 30 h at 37 °C with 5% CO2. The frequency of live cells was determined according to the manufacturer’s protocol (Cat #R37601, Thermo Fisher, USA).
10
Evaluating Cell Viability with Calcein AM
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For viability assays, hscAS on the P/F‐LOCS were treated with 2 μM calcein AM and 4 μM ethidium homodimer‐1 (R37601, Thermo Fisher Scientific) in differentiation medium for 15 min, and then observed under a Leica TCS SP8 confocal microscope (Leica Microsystems). Cell viability was calculated using ImageJ software (n = 3 samples).
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