Arabidopsis thaliana seeds were obtained from the Arabidopsis Biological Resource Center (stock no. CS4004) and placed on a Lloyd & McCown Woody Plant Basal Medium with Vitamins (PhytoTec Labs) agar pad sitting atop a 35 mm glass bottom dish (Thermo Fisher Scientific, 150682). The pad and seeds were incubated at 4 °C for 3 days, then moved to room temperature under lab bench lights for 9 days. Rhobo6 was added at 5 μM to water surrounding the agar pad overnight. Root structures within the agar pad were imaged the following morning.
35 mm glass bottom dish
Manufactured by Thermo Fisher Scientific
The 35 mm glass bottom dish is a laboratory equipment product designed for cell culturing and microscopy applications. It provides a transparent glass surface for growing and observing cells. The dish is made of high-quality materials and is suitable for a variety of experimental setups.
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4 protocols using 35 mm glass bottom dish
1
Imaging Arabidopsis Root Development
2
Caspase-3/7 Assay and WST-8 Metabolic Activity of GNPs
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Caspase-3/7 assay: 0.5×10 5 U251 cells were seeded on a 35 mm glass-bottom dish (ThermoFisher SCIENTIFIC) and incubated at 37°C for 24 h. After 24 h, the culture medium was replaced with fresh medium containing 25 μg/mL GNPs functionalized with/ without cyt c and incubated for 4 h. After 4 h, media containing GNPs was removed and cells were washed three times with PBS. Next, the cells were incubated with 8 µM CellEventTM Caspase 3-7 green detection reagent in PBS containing 5% FBS for 30 min at 37 ºC.
Afterwards, the cells were preserved with 3.7% formaldehyde for 15 min and subsequently washed with PBS. Later the cells were treated with DAPI for 5 min at RT under dark and washed again with PBS. Finally, the cells were immersed in PBS and imaged using Zeiss Elyra 2.9. WST-8 metabolic activity assay: Biocompatibility of GNPs and cyt c bound GNPs was studied using WST-8 assay. A total of 1 × 10 4 U251 cells were incubated on 96 well plates 24 h prior experiment. During the next step, the culture media was replaced with medium containing GNPs (12.5 or 25 µg/mL) and incubated for 4 h. Next, media was replaced with 10% WST-8 in complete DMEM and incubated for an hour before reading the absorbance at 450 nm in a Tecan microplate reader. Culture media and 3% Triton X-100 were taken as negative and positive control respectively. Values are presented relative to negative controls.
Afterwards, the cells were preserved with 3.7% formaldehyde for 15 min and subsequently washed with PBS. Later the cells were treated with DAPI for 5 min at RT under dark and washed again with PBS. Finally, the cells were immersed in PBS and imaged using Zeiss Elyra 2.9. WST-8 metabolic activity assay: Biocompatibility of GNPs and cyt c bound GNPs was studied using WST-8 assay. A total of 1 × 10 4 U251 cells were incubated on 96 well plates 24 h prior experiment. During the next step, the culture media was replaced with medium containing GNPs (12.5 or 25 µg/mL) and incubated for 4 h. Next, media was replaced with 10% WST-8 in complete DMEM and incubated for an hour before reading the absorbance at 450 nm in a Tecan microplate reader. Culture media and 3% Triton X-100 were taken as negative and positive control respectively. Values are presented relative to negative controls.
3
HeLa Cells Transfection and Photobleaching
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HeLa histone eGFP cells
were obtained from ATCC and cultured in Dulbecco’s modified
Eagle’s medium (DMEM) without phenol-red (Gibco Life Technologies,
Invitrogen) supplemented with 10% fetal bovine serum (FBS), 1% glutamax,
and 50 μg/mL gentamicin at 37 °C with 5% CO2. Twenty-four hours before transfection, 2.4 × 105 cells suspended in 3 mL of complete growth medium were seeded in
a 35 mm glass-bottom dish (ThermoFisher Scientific) equipped with
an adhesive silicone isolator (24-well, Grace Bio-Labs) with 4.5 mm
feature well diameter. The pEGFP plasmid was a kind gift from Prof.
Hideaki Mizuno. The vector (50 ng/well) was transfected into cells
with 0.1 μL/well of TransIT-X2 (Mirus Bio) according to the
manufacturer’s instructions. Sixteen hours after transfection,
the cells were washed once with prewarmed phosphate-buffered saline
(PBS) and then fixed with 4% paraformaldehyde in PBS for 15 min at
room temperature. After fixation, the cells were washed twice with
PBS, permeabilized for 15 min using PBS containing 0.1% Triton X-100,
and washed three times for 5 min using PBS before proceeding to photobleaching.
were obtained from ATCC and cultured in Dulbecco’s modified
Eagle’s medium (DMEM) without phenol-red (Gibco Life Technologies,
Invitrogen) supplemented with 10% fetal bovine serum (FBS), 1% glutamax,
and 50 μg/mL gentamicin at 37 °C with 5% CO2. Twenty-four hours before transfection, 2.4 × 105 cells suspended in 3 mL of complete growth medium were seeded in
a 35 mm glass-bottom dish (ThermoFisher Scientific) equipped with
an adhesive silicone isolator (24-well, Grace Bio-Labs) with 4.5 mm
feature well diameter. The pEGFP plasmid was a kind gift from Prof.
Hideaki Mizuno. The vector (50 ng/well) was transfected into cells
with 0.1 μL/well of TransIT-X2 (Mirus Bio) according to the
manufacturer’s instructions. Sixteen hours after transfection,
the cells were washed once with prewarmed phosphate-buffered saline
(PBS) and then fixed with 4% paraformaldehyde in PBS for 15 min at
room temperature. After fixation, the cells were washed twice with
PBS, permeabilized for 15 min using PBS containing 0.1% Triton X-100,
and washed three times for 5 min using PBS before proceeding to photobleaching.
4
Time-lapse Microscopy of Yeast Cells
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Strains JRY12861, JRY12564, and 12901 were grown as described above for flow cytometry, but in 5 mL cultures of CSM. After 24 hours of log-phase growth, a 500 µL aliquot of cell suspension at approximately 0.6-1.0 OD was harvested and resuspended in 500 uL sterile water. This cellular suspension was then sonicated for 5 seconds at 20% amplitude (Branson Ultrasonics Digital Sonifier 100-132-888R with Sonicator Tip 101-135-066R) to disrupt aggregates. A 5 µL aliquot of sonicated cells was spotted onto a CSM 2% agar pad. Once dry, the agar pads were inverted onto a 35 mm glass bottom dish (Thermo Scientific) and imaged using a Zeiss Z1 inverted fluorescence microscope with a Prime 95B sCMOS camera (Teledyne Photometrics), Plan-Apochromat 63x/1.40 oil immersion objective (Zeiss) filters, MS-2000 XYZ automated stage (Applied Scientific Instrumentation), and Micro-Manager imaging software (Open Imaging). Samples were incubated at 30°C and imaged every 10 minutes or 15 minutes for a total of 10 hours in bright-field, GFP, and RFP. Time-lapse movies were prepared and analyzed using FIJI software (Schindelin et al. 2012) (link).
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