Fission yeast cells were preincubated in YEA medium at 32°C and plated at 4.0 × 10 5 cells in the central area of 35 mm glass-base dishes coated with poly-L-lysine (Matsunami Glass). After incubation for 15 min at room temperature, the supernatant was removed, and 100 μL of YEA medium containing 1% (w/v) microwave-heated low-melting point agarose (SeaPlaque agarose, 50101; Lonza) was gently added to the central area. After resting for 15 minutes at room temperature, the cells were observed by time-lapse imaging, and 10 μM m D cTMP4Me in 2 mL YEA was added to the dish.
Poly l lysine (pll)
Manufactured by Matsunami
Sourced in Japan
Poly-L-lysine is a synthetic polymer composed of the amino acid L-lysine. It is a positively charged molecule that can be used to modify surfaces and promote cell adhesion and growth in various cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Fluoromount, by Diagnostic BioSystems (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Collagenase type 2, by Worthington (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Horse serum, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Fujifilm (2 mentions)
100 μm cell strainer, by BD (2 mentions)
Lsm 800, by Zeiss (2 mentions)
Seaplaque agarose, by Lonza (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
35 mm dish, by Iwaki (1 mentions)
4 6 diamidino 2 phenylindole dilactate dapi, by Dojindo Laboratories (1 mentions)
Axio observer microscope, by Zeiss (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Dulbecco s modified eagle medium, by Merck Group (1 mentions)
Nutrient f 12 ham, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
11 protocols using poly l lysine (pll)
1
Fission Yeast Live Cell Imaging
2
Tomato Leaf Nuclei Spread Protocol
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Nuclei of tomato leaves were spread on glass slides for a pilot experiment to determine the optimum temperature for the RGEN-ISL method. The 3.0% PFA-fixed and washed tomato leaves were digested with a mixture of 1.0% (w/v) cellulase Onozuka RS and 0.5% (w/v) pectolyase Y-23 dissolved in PBS, by incubation for 60 min at 37 °C. Nuclei released by the digestion were compressed on to slides coated with poly-l -lysine (Matsunami, Osaka, Japan).
3
Neutrophil-M. bovis Interaction Assay
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Neutrophils (concentration of 1 × 106 cells suspended in 100 μL RPMI medium with 10% FBS) were seeded onto glass coverslips treated with 0.001% poly-l-lysine (Matsunami glass, Tokyo, Japan) and placed in a 35 mm dish (Iwaki, Shizuoka, Japan). Cells were incubated for 1 h at 37 °C in 5% CO2. Neutrophils were incubated with PMA for 30 min to induce NETs formation (or with PBS for control), and then, 107 CFU octadecyl rhodamine B chloride (Sigma-Aldrich Corp.) labeled live or heat-killed M. bovis (or with PBS for control) were added and incubated for 30 min at 37 °C under a 5% CO2. Neutrophils were washed with PBS and stained with 4,6-diamidino-2-phenylindole, dilactate (DAPI) for 15 min (Dojindo, Tokyo, Japan). Coverslips were washed with PBS, coated with Fluoromount (Diagnostic Biosystems, Pleasanton, CA, USA), and viewed using a fluorescence microscope (Nikon, Tokyo, Japan). Three bovine neutrophil studies were performed individually.
4
Neutrophil Response to Streptococcus uberis
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Totally, 1 × 106 neutrophils, suspended in 100 µL Roswell Park Memorial
Institute (RPMI) 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) and 10% FBS, were seeded
onto glass coverslips treated with 0.001% poly-L-lysine (Matsunami glass, Tokyo, Japan)
and incubated in a 35 mm dish for 1 hr at 37°C with 5% CO2. Neutrophils were
incubated with S. uberis (multiplicity of infection (MOI)=1, 10, and 100)
for 30 min at 37°C with 5% CO2, washed using sterile PBS, and stained with DAPI
for 15 min. Coverslips were washed using PBS, coated with Fluoromount (Diagnostic
Biosystems, Pleasanton, CA, USA), and examined using a ZEISS Axio Observer microscope
(Carl Zeiss). This experiment was conducted using three individual bovine neutrophil
samples.
Institute (RPMI) 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) and 10% FBS, were seeded
onto glass coverslips treated with 0.001% poly-L-lysine (Matsunami glass, Tokyo, Japan)
and incubated in a 35 mm dish for 1 hr at 37°C with 5% CO2. Neutrophils were
incubated with S. uberis (multiplicity of infection (MOI)=1, 10, and 100)
for 30 min at 37°C with 5% CO2, washed using sterile PBS, and stained with DAPI
for 15 min. Coverslips were washed using PBS, coated with Fluoromount (Diagnostic
Biosystems, Pleasanton, CA, USA), and examined using a ZEISS Axio Observer microscope
(Carl Zeiss). This experiment was conducted using three individual bovine neutrophil
samples.
5
DRG Neuron Isolation and Culture
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Culture preparation was conducted as previously described (Masuoka et al., 2015 (link), 2016 (link)). The C57BL/6J male and female mice (6- to 14-day-old) were anesthetized by the inhalation of isoflurane (Escain®; Mylan Inc., Cecil Township, PA, USA). DRG were rapidly dissected in ice-cold Ca2+/Mg2+-free artificial cerebrospinal fluid (Ca2+/Mg2+-free ACSF; 143.9 mM NaCl, 3.35 mM KCl, 21 mM NaHCO3, 9.9 mM glucose, 0.6 mM NaH2PO4) gassed with a mixture of 95% O2 and 5% CO2 (pH 7.4). Neurons were dissociated following treatment with 0.1% type II collagenase (240–265 U/mg; Worthington Biochemical Co., Lakewood, NJ, USA), 0.1% trypsin (Gibco, San Diego, CA, USA), and 0.01% DNase I (Sigma, St. Louis, MO, USA) in Ca2+/Mg2+-free ACSF and shaken (35 cycle/min) in a water bath at 37°C for 30 min. Cells were gently triturated in Dulbecco's modified Eagle medium (Sigma) containing 10% horse serum (Gibco), 5% fetal calf serum (Gibco), and 1% penicillin–streptomycin (Wako, Osaka, Japan). Dispersed cells were passed through a 100-μm cell strainer (BD Biosciences, San Jose, CA, USA), and the filtered cells were seeded on glass coverslips (13 mm in diameter) coated with poly-L-lysine (Matsunami Glass Ind., Osaka, Japan).
6
Visualization of FAM-labeled Peptides in CHO-K1 Cells
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CLSM observation via the z-stack imaging mode was performed on an LSM 800 (Carl Zeiss, Oberkochen, Germany) with a C-Apochromat 40×/1.20 W Korr objective at an excitation wavelength of 488 nm for the visualization of FAM-labeled peptides as described11 (link). CHO-K1 cells (2 × 105 cells) were plated in a 35-mm glass-bottom dish coated with poly-L-lysine (Matsunami Glass Ind. Ltd., Osaka, Japan) and were incubated in nutrient F-12 Ham (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Lonza Group, Basel, Switzerland). After incubation for 24 h (37 °C, 5% CO2), the cells were incubated with FAM-labeled peptides for 30 min at 4 or 37 °C in FBS-free F12-Ham medium. After the incubation, the cells were washed thrice with phosphate-buffered saline (PBS) on ice and stored in PBS, followed by confocal microscopic imaging. The nuclei of cells were counterstained with Hoechst 33342 (Life Technologies, Waltham, MA, USA), following the manufacturer's instructions, and visualized at an excitation wavelength of 405 nm. Throughout image acquisition, laser intensity, photomultiplier detector sensitivity and pinhole aperture values were kept constant.
7
Peptide Localization in Cells by CLSM
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CLSM observation by z-stack imaging mode was performed on a LSM 800 (Carl Zeiss, Oberkochen, Germany) with a C-Apochromat 40×/1.20 W Korr objective at an excitation wavelength of 488 nm (Ar laser) for the visualization of FAM-labeled peptide [11] . To visualize cell morphology, we obtained the CLSM images with differential interference contrast (DIC) images. Cells (2.0 × 10 5 cells) were plated in 35 mm glass-bottom dishes coated with poly-L-lysine (Matsunami Glass Ind. Ltd., Osaka, Japan) and were incubated in growth medium. After incubation for 24 h (37 °C, 5% CO 2 ), the cells were incubated with 10 μM of FAM-labeled peptides for 30 min at 4 °C or 37 °C in FBS-free F12-Ham medium. After the incubation, the cells were washed 3 times with phosphate-buffered saline (PBS) on ice, and kept in PBS, followed by confocal microscopic imaging. For heparin washing experiments, after the PBS washing, the cells were washed twice with PBS containing 100 μg/mL heparin, washed 3 times with PBS, and kept in PBS, followed by confocal microscopic imaging. The nuclei were labeled with Hoechst 33342 (Life Technologies, Waltham, MA) by following the manufacturer's instructions.
8
Localization of Fluorescent Liposomes in Macrophages
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The localization of the NBD-labeled liposomes was determined using a confocal microscope (LCM710; Zeiss, Oberkochen, Germany). Data were collected using the Zen software (2012). THP-1 cells (2 × 10 5 cells per well) were plated in 35-mm glass-bottom dishes coated with poly-L-lysine (Matsunami, Osaka, Japan), each containing 1.5 mL of RPMI-1640 supplemented with 10% FBS, and were differentiated with PMA into macrophages, as described above. After incubation for 48 h (37 °C, 5% CO 2 ), cells were exposed to the fluorescent-labeled liposomes, DPPC/Chol (80/20 mol%) and DPPC/Chol (67/33 mol%) (final lipid concentration was approximately 50 µg/mL, adjusted to ensure that the two liposome solutions had equal fluorescence intensity before addition to cells). After overnight incubation, the cells were washed twice with HBSS, late endosomes/lysosomes were labeled with LysoTracker RED DND-99 (Thermo Fisher Scientific), and nuclei were labeled with Hoechst 33342 (Thermo Fisher Scientific).
9
Mandibular Molar Histomorphometric Analysis
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The mandibles were dissected and most soft tissue was removed. The specimens were immediately immersed in 10% buffered formalin (Wako Pure Chemical Ind. Ltd.; pH 7.0) at 4℃ overnight and decalcified in 10% ethylenediaminetetraacetic acid (EDTA) at 4℃ for 6 weeks. They were embedded in paraffin and cut into 5-µm-thick sagittal sections parallel to the long axis of the roots of the mandibular first molar.8 ,10 (link) The sections were finally mounted on glass slides coated with poly-L-lysine (Matsunami Glass Ind. Ltd., Osaka, Japan) and stained with hematoxylin and eosin for histomorphometric examination. The observational area included the dental pulp, PDL, alveolar bone proper, and inter-radicular alveolar bone (Figure 1B ).
10
Immunofluorescence Analysis of ACE2 Expression in A549 Cells
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A549 cells grown on cover slides pretreated with poly-l -lysine (Matsunami Glass, Osaka, Japan) were treated with CSF for 48 h. The slides were washed with PBS and then fixed with 4% paraformaldehyde (Wako Pure Chemical Industries, Osaka, Japan) for 15 min at room temperature. The slides were blocked with 2% bovine serum albumin (BSA) for 1 h and incubated with anti-ACE2 antibody (R&D Systems, Minneapolis, MN, USA) in blocking buffer overnight at 4 °C. After thorough rinsing with PBS, the slides were incubated with Alexa Fluor 555 anti-goat secondary antibody (Abcam, Cambridge, MA, USA) in PBS containing 2% BSA and Hoechst 33342 (Thermo Fisher Scientific). The cover slides were mounted on glass slides using the ProLong Gold antifade reagent (Thermo Fisher Scientific) and examined using a fluorescence microscope (IX-FLA, Olympus, Tokyo, Japan). The images obtained were subsequently analyzed using InStudio (Pixera, Santa Clara, CA, USA).
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