For siRNA transfection, HeLa cells were plated in six-well plates (Thermo Scientific) at 0.8×105 cells per well and transfected the following day with control siRNA (Thermo Scientific AM4635) or predesigned SEPT7 siRNA (Thermo Scientific s2753) using Oligofectamine (Thermo Scientific) for 72 h. For DNA transfection, 5×105 HeLa cells were seeded per MatTek glass-bottom dish (MatTek corporation) including DNA and JetPEI (Polyplus transfection), and were used 24 h later.
Glass bottom dish
Manufactured by MatTek
Sourced in United States, Japan
The glass-bottom dish is a laboratory equipment item that provides a transparent glass substrate for cell culture and microscopy applications. It allows for direct observation and imaging of cells or samples placed within the dish.
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Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Lsm 880, by Zeiss (4 mentions)
Lsm 780, by Zeiss (4 mentions)
Daq board, by Nikon (3 mentions)
Nis element, by Nikon (3 mentions)
A1rmp two photon microscope, by Nikon (3 mentions)
Ultraview vox, by PerkinElmer (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Eclipse ti inverted microscope, by Nikon (2 mentions)
Primovert microscope, by Zeiss (2 mentions)
Eclipse ti2 e inverted microscope, by Nikon (2 mentions)
Lsm 800 confocal laser scanning microscope, by Zeiss (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Live cell environmental chamber, by Tokai Hit (2 mentions)
Cytochalasin d, by Merck Group (2 mentions)
Fetal calf serum (fcs), by Merck Group (2 mentions)
109 protocols using glass bottom dish
1
siRNA and DNA Transfection Protocol for HeLa Cells
2
STORM Imaging of Motile Sperm
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For STORM, motile sperm were selected by either density gradient or the swim-up technique. The swim-up technique for STORM was performed in two different buffers, wash buffer (PSW-100) or modified Krebs–Ringer Hepes (mKRH) medium (pH 7.4, lacking CaCl2, NaHCO3, and Bovine Serum Albumin) composed of 94.0 mM NaCl, 1.19 mM MgSO4·7H2O, 1.19 mM KH2PO4, 4.78 mM KCl, 25.07 mM HEPES, 27.64 mM glucose, 50 mg/ml streptomycin sulfate and 100 IU/ml penicillin G potassium salt. Motile sperm were placed on round cover glass (Fisher Scientific, 72231-01) or MatTek glass-bottom dish (MatTeK corporation, p35G-1.5-10.C), allowed 5 min for movement, and snap-frozen in liquid nitrogen. The snap-frozen sperm were fixed in prechilled methanol for 3 min and prepared for immunostaining as described in the “Immunofluorescence” section.
3
Measuring Protein Turnover in Live Cells
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MCF‐7 + p53‐Venus + MDM2p‐mCherry‐NLS‐PEST cells were plated into a 6‐well MatTek glass bottom dish (MatTek corporation) and treated with 10 μM Nutlin‐3 overnight to induce mCherry expression. Following the overnight induction, Nutlin‐3 was removed by washing cells three times with Transparent RPMI media containing 5% FBS and 1% antibiotic–antimycotic solution. Cells were then treated with cycloheximide (100 μg/ml) in Transparent RPMI media and immediately imaged for 24 h by time‐lapse fluorescence microscopy. Images were analyzed using ImageJ, and customized MATLAB code was used to determine the half‐life of the mCherry reporter protein.
4
Live Imaging of Vaccinia Virus Actin Tails
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HeLa cells (American Type Culture Collection, Manassas, VA) were grown in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS; Life Technologies) at 37°C in a 5% CO2 incubator. Cells were seeded on glass coverslips at 20% confluency, infected with vB5R-GFP vaccinia virus (Ward and Moss, 2001 (link)) at 1 plaque-forming unit (pfu)/cell for 1 h, and then washed three times with PBS before addition of fresh medium. At 12 h after infection, cells were washed three times in PBS, fixed in 4% paraformaldehyde in PBS, permeabilized with 0.05% Triton X-100 in PBS, and stained with phalloidin–Alexa Fluor 568 (Life Technologies) to visualize actin tails. For time-lapse video microscopy (Alvarez and Agaisse, 2013 (link)), cells were seeded on a 35-mm MatTek glass-bottom dish (MatTek) at a confluency of 30–50%. Cells were transfected with pYFP–N-WASP and actin-CFP the day before infection and infected with WR vaccinia virus (Ward and Moss, 2001 (link)) at 10 pfu/cell. Images were captured 10 h after infection every 12 s on a spinning-disk confocal microscope (TE2000E) using a 60× oil objective with Volocity software (PerkinElmer). See Supplemental Figures S4 and S6B and Supplemental Movie S6.
5
Live Imaging of Drosophila Embryos
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To prepare embryos for live imaging, manually staged embryos expressing anillin-GFP/ sqh-mCherry or sqh-GFP were collected at room temperature (22-25°C) on agar plates, dechorionated in 50% bleach for 1-2 min, rinsed thoroughly with water, and transferred on a 35 mm MatTek glass-bottom dish (MatTek Corporation). Distilled water was then added to the dish well to completely cover the embryos. All live imaging was performed at room temperature on a Nikon inverted spinning disk confocal microscope equipped with Andor W1 dual camera, dual spinning disk module. A CFI Plan Apo Lambda 60×/1.40 WD 0.13 mm Oil Objective Lens objective was used for imaging. GFP-and mCherry-tagged proteins were imaged with a 488-nm laser and a 561-nm laser, respectively.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
6
Imaging Expanded Plant Samples
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Expanded samples were placed in a MatTek glass-bottom dish. Excess water around the gel was wicked off with filter paper. To maintain humidity in the chamber, wet filter paper strips were plastered against the inner wall of the dish and the dish remained covered throughout imaging. Wide-field image stacks were collected at z-increments of 0.3 μm using a DeltaVision OMX Flex imaging station with a 60x oil immersion lens (NA 1.42) and immersion oil at refractive index 1.520. Images were deconvolved using Softworx (GE Healthcare-Applied Precision) with the point spread function supplied by the manufacturer. Contrast levels were adjusted to optimize the display.
7
Live-Cell and Fixed-Cell Imaging
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For live-cell microscopy, cells transfected with interested DNA constructs were seeded on glass-bottom dish (Mattek) and imaged with Zeiss inverted microscopy Axio Observer Z1 at 37°C with 63×, 1.35-NA oil-immersion objective. To image fixed cells, sample slides were observed with confocal microscopy LSM700 with 63×, 1.35-NA oil-immersion objective (Carl Zeiss, Jena, Germany).
8
Immunofluorescence Staining of IZUMO1 Expressing Cells
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Regarding live cell surface staining, IZUMO1_v1 or v2-expressing COS-7 cells on the glass-bottom dish (MatTek Corporation) were washed with PBS, and incubated with 0.5 μg/ml anti-IZUMO1 antibody (Mab18-Alexa546) and 1 μg/ml Hoechst 33342 (Sigma-Aldrich) for 2 hours at 37 °C. As for cytoplasmic staining, IZUMO1_v1 or v2-expressing COS-7 cells were incubated with 500 nM MitoTracker Red CMXRos (Thermo Fisher Scientific) at 37 °C for 30 min and fixed in 1% paraformaldehyde/PBS for 1 hour, followed by incubation with 0.2% Triton-X100/PBS for 10 min. After washing three times with PBS, fixed cells were covered with blocking buffer (1% BSA in PBS) for 30 min and incubated with 0.5 μg/ml anti-IZUMO1 antibody (Mab18-Alexa488) and 1 μg/ml Hoechst 33342 for 1 hour at 37 °C. After labelling, the cells were washed and observed under an A1R confocal microscope (Nikon) with a 40 × objective (NA 0.95).
9
Microscopic Analysis of B. subtilis
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Microscopy was completed by taking 1-ml aliquots of B. subtilis cultures and washing with 1× phosphate-buffered saline (PBS) through centrifugation. Cells were then resuspended in 100 μl of PBS, and the red membrane stain FM4-64 was added at a final concentration of 1 μg/ml. The sample was prepared for microscopy by spotting 5 μl of the cell suspension onto the glass coverslip of a MatTek glass bottom dish and then covering it with a 1% agarose pad made with sterile water, as described previously (23 (link)). All imaging was completed at room temperature inside an environmental chamber using a GE Applied Precision DeltaVision Elite deconvolution fluorescence microscope. Photos were taken using a Photometrics CoolSnap HQ2 camera. All images were acquired by taking 17 z-stacks at 200-nm intervals. Images were deconvolved though the SoftWorx imaging software provided by the microscope manufacturer.
10
Live-Cell Imaging of NK Cells
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For live-cell imaging, carboxyfluorescein diacetate succinmidyl ester (CFSE)-labeled HLCZ01 or primary NK cells were plated on a glass-bottom dish (MatTek, Bratislave, Slovak Republic). The live-cell confocal time-lapse sequences were taken on a Zeiss Cell Observer s.d. Confocal Microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). Excitation wavelengths of 488 and 639 nm were selected for CFSE (Beyotime, Nanjing, China) and DiD, respectively. Emission was detected by a 60 × or 100 × oil-immersion objective, and the images were collected in a single z-plane. A stage-top incubator was set to 37 °C and 5% CO2 to keep the cells alive. All the images were acquired and processed with the Zeiss AxioVision software package (Carl Zeiss Microscopy GmbH).
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