Fluorescein diacetate (FDA) and propidium iodide (PI) nucleic acid staining dyes (Molecular Probes, Inc., Eugene, OR, USA) were used to examine the viable and dead populations as described in our previous study (Xiao et al., 2022 (link)). FDA can penetrate both viable and damaged cells, while PI only intercalates into the DNA of damaged cells. The stained cells were determined by using Guava easyCyte™ 8-8HT flow cytometer (Guava Technologies Inc., Hayward, CA, USA). The viable cells emitting green fluorescence and dead cells emitting red fluorescence were counted, respectively, and total yeast count is the sum of the numbers of viable cells and dead cells. In addition, the conventional YPD plate culture was carried out for counting of culturable cells as described elsewhere (Deng et al., 2018 (link)). The difference between the viable and culturable cell counts was identified as the number of VBNC cells.
Fluorescein diacetate fda
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Fluorescein diacetate (FDA) is a fluorescent dye used in various laboratory applications. It functions as a cell viability indicator, enabling the detection and quantification of living cells. FDA is a non-fluorescent, lipophilic compound that can passively diffuse into cells. Once inside, cellular esterases cleave the acetate groups, releasing the fluorescent compound fluorescein, which can then be detected using appropriate instrumentation.
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Lab products found in correlation
Propidium iodide, by Thermo Fisher Scientific (9 mentions)
Calcein am, by Thermo Fisher Scientific (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Rhodamine b, by Merck Group (1 mentions)
Collagenase 4, by Merck Group (1 mentions)
Sp5 laser scanning confocal microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Acyclovir acv, by Merck Group (1 mentions)
3.5 cm petri dishes, by Corning (1 mentions)
Nelfinavir, by Merck Group (1 mentions)
Benzbromarone, by Merck Group (1 mentions)
Riddelliine, by Phytolab (1 mentions)
Lasiocarpine, by Phytolab (1 mentions)
Cyclophosphamide, by Thermo Fisher Scientific (1 mentions)
Vincristine, by Merck Group (1 mentions)
Quinidine, by Merck Group (1 mentions)
Ketoconazole, by Merck Group (1 mentions)
20 protocols using fluorescein diacetate fda
1
Quantifying Viable and Dead Yeast Cells
2
Primary Rat Hepatocyte Cultivation
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Male SD rats aged 5–8 weeks old (Sichuan Dashuo Biotech Inc., Chengdu, China) were anaesthetized. All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Sichuan Agricultural University and were in accordance with the Guide for the Care and Use of Laboratory Animals. Primary hepatocytes were isolated from the livers by a two-step in situ collagenase perfusion method. The viability of hepatocytes was determined by the trypan blue exclusion assay, and a viability of >90% was used. Fluorescein diacetate (FDA) were purchased from Molecular Probes (Carlsbad, CA, USA). The microfluidic channel was sterilized with 75% ethanol for 30 min prior to use. Hepatocytes were seeded at a density of 5 × 106 cells per well on the various substrata. After cells attached for 10 h under a static environment, DMEM with 10% (v/v) fetal bovine serum was perfused into the microchannel at various flow rates. The microfluidics-based cell-culture platform was then placed in an incubator with 5% CO2 at 37 °C for 15 days.
3
Synthesis and Characterization of PLA-PDMS Composite
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Poly-dl -lactide (PLA, MW = 180 kDa) was synthesized in our laboratory. PDMS (Sylgard 184 Silicone kit) was purchased from Dow Corning (Midland, MI, USA). Rhodamine B and collagenase IV were procured from Sigma Aldrich (St. Louis, MO, USA). Calcein-AM, propidium iodide, and fluorescein diacetate (FDA) were purchased from Molecular Probes (Carlsbad, CA, USA). Anti-goat IgG–FITC (fluorescein isothiocyanate) was purchased from Biosynthesis Biotechnology Co., Ltd. (Beijing, China). Ag NPs (particle size 20–40 nm) were purchased from Nanonasb-Pars Company (Tehran, Iran). All chemical reagents used were of analytical grade and obtained from Chengdu Kelong Reagent Co. (Chengdu, China) unless otherwise indicated.
4
Cell Viability and Cytoskeleton Imaging
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The in vitro tissue-engineered construct was stained with 2 µg/mL fluorescein diacetate (FDA) and 20 µg/mL propidium iodide (PI; both from Life Technologies) to detect living and dead cells, respectively. Additional constructs were fixed overnight at 4 °C in 4% paraformaldehyde (Sigma) then stained with 0.3 U/mL rhodamine 415-conjugated phalloidin and 2.5 µg/mL 4′6-diamidino-2-phenylindole (DAPI; Life Technologies). Samples were imaged with a Leica SP5 confocal scanning laser microscope49 (link).
5
Fluorescent Vital Staining Microscopy
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Vital staining was performed with 5 μg/ml fluorescein diacetate (FDA) (Life Technologies, Carlsbad, California, USA) and 20 μg/ml propidium iodide (PI) (Life Technologies) in PBS on cells incubated in the dark for 5 min at room temperature. All images were observed and photographed using a laser scanning confocal microscope (Nikon A1, Tokyo, Japan).
6
Antiviral Efficacy of Indolicidin and LL-37 in HSV-1 Infection
7
Islet Viability Assessment via Fluorescence
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After 24 h in culture, purified islets were handpicked and placed into 3.5 cm petri dishes (Corning, USA) containing 3 ml of culture media.
Islet viability (n=20) was assessed by double fluorescence staining with 20 μl propidium iodide (PI 1:10) followed by 20 μl of fluorescein diacetate (FDA; 1:100; Life Technologies, Foster city, USA) in 3 ml culture media for 30 seconds and then washed in PBS. The average viability of each islet was assessed with inverted wide field fluorescence microscope (Axiovert 200M, Zeiss, Feldbach, Switzerland) on five separate fields based on the percentage of viable versus dead cells.
Islet viability (n=20) was assessed by double fluorescence staining with 20 μl propidium iodide (PI 1:10) followed by 20 μl of fluorescein diacetate (FDA; 1:100; Life Technologies, Foster city, USA) in 3 ml culture media for 30 seconds and then washed in PBS. The average viability of each islet was assessed with inverted wide field fluorescence microscope (Axiovert 200M, Zeiss, Feldbach, Switzerland) on five separate fields based on the percentage of viable versus dead cells.
8
Evaluation of Cell Cytotoxicity Assays
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PAs (europine (CAS# 570-19-4; assay 100%), riddelliine (CAS# 23,246-96-0; assay ≥ 98%) and lasiocarpine (CAS# 303-34-4; assay 100%) were obtained from PhytoLab (Vestenbergsgreuth, Bayern, Germany); Cyclophosphamide (CAS# 6055–19-2; assay ≥ 97%) was from Alfa Aesar (Karlsruhe, Germany). Gel Green Nucleic Acid stain was obtained from Biotium (Darmstadt, Germany). Fluorescein diacetate (FDA; CAS# 596-09-8) was from Invitrogen (Germany). Dimethylsulfoximide (DMSO; ≥ 99.8%), bisBenzimide H33258 (CAS# 23,491–45-4; assay ≥ 98%), diazabicyclo-octane (DABCO), ketoconazole (CAS# 65,277-42-1; assay ≥ 98%), quinidine (CAS# 56-54-2; assay ≥ 97%), verapamil (CAS# 152-11-4; assay ≥ 99%), nelfinavir (CAS# 159,989-65-8; assay ≥ 98%), benzbromarone (CAS# 3562-84-3; assay ≥ 95%), vincristine (CAS# 2068-78-2; assay ≥ 97%), cytochalasin B (CAS# 14,930-96-2), ethidium bromide (CAS# 1239-45-8; assay ≥ 95%) and sodium fluorescein (CAS# 518-47-8; assay ≥ 95%) were from Sigma-Aldrich (Steinheim, Germany). Calcein-acetoxymethyl ester (Calcein-AM; CAS# 148,504-34-1; assay ≥ 95%) was from Cayman Chemical Company (Germany). Cell culture media and reagents were all from Sigma-Aldrich (Steinheim, Germany), except foetal bovine serum, which was from Biochrom (Berlin, Germany).
9
Analyzing Microalgal Responses to Stress
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Carboys of stressed and control CC and T- Iso were fed to the oyster larvae when the algal cultures were 5 days old and 7 days old, respectively. On T0, T24 h, T48 h, and T72 h of the larval exposure assay, approximately 50 mL from non-stressed/control and stressed cultures of CC and T-Iso were collected before feeding the larvae to determine a) pH, b) microalgal cell concentration, c) photosynthetic, and d) physiological parameters (Figure 1 ).
Cellular assays, extensively described in Rolton et al. (2020) , assessed the: relative size (Forward Scatter, FSC) and complexity (Side Scatter, SSC) of the algae, the chlorophyll content (red fluorescence, FL3), cell viability (SYTOXTM Green, S7020, Invitrogen for CC, and Fluorescein diacetate FDA, F1303, Invitrogen for T-Iso), reactive oxygen species (ROS) production (DCFH-DA, CAS No. 4091-99-0, Sigma Aldrich®) and neutral lipid content (BODIPYTM 493/503, D3922, Molecular probes). Data were analyzed using ‘Cell Quest Pro’ software.
Cellular assays, extensively described in Rolton et al. (2020) , assessed the: relative size (Forward Scatter, FSC) and complexity (Side Scatter, SSC) of the algae, the chlorophyll content (red fluorescence, FL3), cell viability (SYTOXTM Green, S7020, Invitrogen for CC, and Fluorescein diacetate FDA, F1303, Invitrogen for T-Iso), reactive oxygen species (ROS) production (DCFH-DA, CAS No. 4091-99-0, Sigma Aldrich®) and neutral lipid content (BODIPYTM 493/503, D3922, Molecular probes). Data were analyzed using ‘Cell Quest Pro’ software.
10
Cell Line Maintenance and Characterization
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PC-3 (ATCC® CRL-1435) prostate, MDA-MB-453 (ATCC® HTB-131) breast, SKOV-3 (ATCC® HTB-77) ovarian, U87MG (ATCC® HTB-14) glioma, SW900 (ATCC® HTB-59) lung, Ramos (RA1) (ATCC® CRL-1596) lymphoma, and KG1a (ATCC® CCL-246.1) leukemia cell line were purchased from American Type Culture Collection (Manassas, VA, USA). The human head and neck carcinoma cell line scc-U8 was kindly provided by Dr. Robinson (Erasmus MC, Rotterdam, The Netherlands). The cells were cultured in either RPMI 1640 or DMEM medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were cultured at 37 °C in 5% CO2. The water-soluble phthalocyanine dye IRDye 700DX NHS ester was purchased from LI-COR Bioscience (Lincoln, NE, USA). FITC-conjugated anti-Human IgG (Fc specific) antibody was purchased from Sigma (St. Louis, MO, USA), anti-HSP70 antibody (1H1) was purchased from Stress Marq (Biosciences, Cadboro Bay, BC, Canada, c), and anti-Calreticulin antibody (FMC 75) was purchased from Abcam (Cambridge, UK). Propidium iodide, fluorescein diacetate (FDA), and 2′,7′-dichlorodihydro-fluorescein diacetate (H2DCF-DA) were purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals were of reagent grade.
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