COS-7 cells were cultured and transfected as described above. FRAP experiments were performed using a Zeiss LSM880 confocal microscope (PeCon GmbH, Erbach, Germany) and processed with Zen software. FLAP images were obtained using an Olympus FV1200 laser scanning confocal microscope (Olympus, Tokyo, Japan) and analyzed in FV10-ASW2.0 and OriginPro8.
Lsm 880 confocal microscope
Manufactured by Pecon
Sourced in Germany
The LSM 880 is a confocal microscope. It is designed to capture high-resolution images of samples by scanning them with a focused laser beam and detecting the reflected light. The LSM 880 allows for optical sectioning, enabling the visualization of thin slices within a specimen.
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Lab products found in correlation
3 protocols using lsm 880 confocal microscope
1
FRAP and FLAP Imaging of COS-7 Cells
2
Cellular Uptake and Fluorescence Imaging
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For cellular uptake experiments and posterior observation under the microscope, cells were seeded on glass bottom dishes (P35G-1.5-14-C, Mattek). 24 h after cell seeding, cells were incubated at 37 °C for 30 min with COUPY (1 μM) and Ru-COUPY (10 μM) in supplemented DMEM. Then, cells were washed three times with DPBS (Dulbecco's phosphate-buffered saline) to remove the excess of the compounds and kept in DMEM with Hepes (10 mM) and without phenol red for fluorescence imaging. All microscopy observations were performed using a Zeiss LSM 880 confocal microscope equipped with a heating insert P S (Pecon) and using a 63×1.4 oil immersion objective. The compounds were excited using the 633 nm laser and detected from 650 to 750 nm. Image analysis was performed using Fiji.90 (link) Unless otherwise stated images are colorized using Fire lookup table.
3
Cellular Uptake of Encapsulated Coumarin
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HeLa cells were maintained
in DMEM (Dulbecco’s modified
Eagle medium) containing high glucose (4.5 g/L) and were supplemented
with 10% fetal bovine serum (FBS), 50 U/mL penicillin–streptomycin,
and 2 mMl -glutamine. For cellular uptake experiments and
posterior observation under the microscope, cells were seeded on glass
bottom dishes (P35G-1.5-14-C, Mattek). Twenty-four hours after cell
seeding, cells were incubated at 37 °C for 30 min with free and
encapsulated coumarin (1 μM) in supplemented DMEM. To determine
the internalization mechanism of both compounds, low-temperature incubations
were performed at 4 °C during 30 min in the same biological medium
and at the same concentration (1 μM). Then, cells were washed
three times with DPBS (Dulbecco’s phosphate-buffered saline)
to remove the excess of the compounds and kept in low glucose DMEM
without phenol red supplemented with Hepes 10 mM for fluorescence
imaging.
All microscopy observations were performed using a
Zeiss LSM 880 confocal microscope equipped with a heating insert (P
S1, Pecon). In the case of low-temperature internalization, cells
were kept at RT. Cells were observed using a 63× 1.4 oil immersion
objective. The compounds were excited using the 561 nm laser and detected
from 570 to 670 nm. Image analysis was performed using Fiji.35 (link) Unless otherwise stated, images are colorized
using a Fire lookup table.
in DMEM (Dulbecco’s modified
Eagle medium) containing high glucose (4.5 g/L) and were supplemented
with 10% fetal bovine serum (FBS), 50 U/mL penicillin–streptomycin,
and 2 mM
posterior observation under the microscope, cells were seeded on glass
bottom dishes (P35G-1.5-14-C, Mattek). Twenty-four hours after cell
seeding, cells were incubated at 37 °C for 30 min with free and
encapsulated coumarin (1 μM) in supplemented DMEM. To determine
the internalization mechanism of both compounds, low-temperature incubations
were performed at 4 °C during 30 min in the same biological medium
and at the same concentration (1 μM). Then, cells were washed
three times with DPBS (Dulbecco’s phosphate-buffered saline)
to remove the excess of the compounds and kept in low glucose DMEM
without phenol red supplemented with Hepes 10 mM for fluorescence
imaging.
All microscopy observations were performed using a
Zeiss LSM 880 confocal microscope equipped with a heating insert (P
S1, Pecon). In the case of low-temperature internalization, cells
were kept at RT. Cells were observed using a 63× 1.4 oil immersion
objective. The compounds were excited using the 561 nm laser and detected
from 570 to 670 nm. Image analysis was performed using Fiji.35 (link) Unless otherwise stated, images are colorized
using a Fire lookup table.
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