After induction for 14 days, 5 × 104 cells in 500 μl of EGM2 were plated onto 4-well Culture Slide (BD Biosciences, San Jose, CA, USA) that has been pre-coated with 150 μl of growth factor-reduced Matrigel (BD Biosciences) per well. Sixteen hours later, the development of capillary-like networks was examined by phase-contrast microscopy. Tube formation was defined as four times in length than its width. Numbers of tubular branches were counted in 10 random fields per well (Image-Pro Plus) as previously reported [3 (link)].
4 well culture slide
Manufactured by BD
Sourced in United States
The 4-well culture slides are a type of laboratory equipment used for cell culture applications. They provide a containment system with four individual wells for the culturing of cells in a controlled environment.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (2 mentions)
Axioskop 2 plus, by Zeiss (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Optiphot 2, by Nikon (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Zinc formalin, by Merck Group (1 mentions)
Low gelling temperature agarose, by Merck Group (1 mentions)
Deltavision ultra microscope, by GE Healthcare (1 mentions)
Huygens professional version 17, by Scientific Volume Imaging (1 mentions)
Normal goat serum (ngs), by Cell Signaling Technology (1 mentions)
Anti p53 antibody do 1, by Santa Cruz Biotechnology (1 mentions)
Triton x 100, by Promega (1 mentions)
Tyramide signal amplification kit, by PerkinElmer (1 mentions)
Goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Mtt cell proliferation assay kit, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Polarstar galaxy, by BMG Labtech (1 mentions)
Lsm 710 meta, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole dapi containing mounting medium, by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
15 protocols using 4 well culture slide
1
In Vitro Angiogenesis Assay
2
Fluorescence Microscopy Cell Staining Protocol
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Cells (1 mL aliquots) obtained
from a diluted cell suspension were seeded into each well (1.7 cm2, ∼1000 cells/well) of a 4-well culture slide (BD Biosciences)
and grown to confluence in 5% CO2 at 37 °C. After
aspirating the old media, 1 mL of the compound or PS (0.5 μM)
in fresh prewarmed media at 37 °C was added to each well. After
PS treatment for 24 h, cells were washed with 1 mL fresh growth media,
and then irradiated with light using LumaCare LC-122 as described
above. Cells were stained in the dark with 1 mL of 0.1 mg/mL Hoechst
33258 (Molecular Probes) in prewarmed media for 15 min at 37 °C,
washed twice with 1 mL filtered PBS, then fixed with 1 mL filtered
prewarmed 4% paraformaldehyde for 10 min in the incubator. Wells were
removed after liquid aspiration and slides were allowed to air dry
in the dark for 1 h. Slides were then protected with coverslips, sealed
with nail polish, and allowed to dry in the dark for another 30 min.
Images were recorded by fluorescence microscopy [4′,6-diamidino-2-phenylindole
(DAPI) for Hoechst 350–390 nm excitation and 460–490
nm emission filters] using an upright fluorescence microscope with
a Retiga imaging 2000R (Nikon Optiphot-2, 20× and 40×) and
image processing Nikon NIS-Elements V4.0 Qimaging software.
from a diluted cell suspension were seeded into each well (1.7 cm2, ∼1000 cells/well) of a 4-well culture slide (BD Biosciences)
and grown to confluence in 5% CO2 at 37 °C. After
aspirating the old media, 1 mL of the compound or PS (0.5 μM)
in fresh prewarmed media at 37 °C was added to each well. After
PS treatment for 24 h, cells were washed with 1 mL fresh growth media,
and then irradiated with light using LumaCare LC-122 as described
above. Cells were stained in the dark with 1 mL of 0.1 mg/mL Hoechst
33258 (Molecular Probes) in prewarmed media for 15 min at 37 °C,
washed twice with 1 mL filtered PBS, then fixed with 1 mL filtered
prewarmed 4% paraformaldehyde for 10 min in the incubator. Wells were
removed after liquid aspiration and slides were allowed to air dry
in the dark for 1 h. Slides were then protected with coverslips, sealed
with nail polish, and allowed to dry in the dark for another 30 min.
Images were recorded by fluorescence microscopy [4′,6-diamidino-2-phenylindole
(DAPI) for Hoechst 350–390 nm excitation and 460–490
nm emission filters] using an upright fluorescence microscope with
a Retiga imaging 2000R (Nikon Optiphot-2, 20× and 40×) and
image processing Nikon NIS-Elements V4.0 Qimaging software.
3
Pluripotency and β-cell Differentiation Imaging
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For pluripotency test, cell lines were cultured in 4-Well Culture Slide (Falcon) until 70% confluence was reached, then fixed with 4% paraformaldehyde. Terminally differentiated β cell aggregates were embedded in 2–4% of low gelling temperature agarose (Sigma-Aldrich), fixed with 10% zinc formalin (Sigma-Aldrich), then included in paraffin. For intracellular staining cells were permeabilized (PermWash 0.4% Triton X-100 in PBS) and stained by using the antibodies listed in the key resources table . All images were acquired using Confocal UltraVIEW ERS microscope (PerkinElmer Life Sciences) and DeltaVision Ultra microscope (GE Healthcare Life Sciences), then analyzed by using Fiji/ImageJ v.1.52p.
4
Melanocyte Culture on Collagen Surface
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Two wells of a 4-well culture slide (BD Falcon, Franklin Lakes, NJ, USA; #354104) were coated with collagen (Stem Cell Technologies, Vancouver, BC, Canada; #04902) and 3 × 104 cells were plated per well. The cells were grown on the surface of the slide or on the collagen, and in either Melanocyte Growth Medium (ZenBio, Inc., Durham, NC, USA) or WIT-P. After 3 days, the cells were fixed in 4% paraformaldehyde. Anti-MART-1 IHC was carried out as given in Additional file 1 : Table S2.
5
Immunofluorescence Imaging of Human iPSC-derived Cells
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For immunofluorescence human iPSC were differentiated in 4-Well Culture Slide
(Falcon, Corning). HI were embedded in agarose, fixed with 4% paraformaldehyde,
included in paraffin and 3 μm sections were cut with a microtome. Sections were
de-paraffinized using xylene, and rehydrated in a serial dilution of absolute
alcohol. Cells were permeabilized when necessary for intracellular staining
(PermWash 0.2% Triton X-100 in PBS) and stained using the antibodies listed in
Tables 1 and
2 . Images were
acquired using Confocals PerkinElmer UltraVIEW ERS microscope (PerkinElmer Life
Sciences, Waltham, MA, USA) and deconvolved with Huygens Professional version
17.04 (Scientific Volume Imaging), using the Classic Maximum Likelihood
Estimation (CMLE) algorithm, with signal-to-noise ratio (SNR): 100
iterations.
(Falcon, Corning). HI were embedded in agarose, fixed with 4% paraformaldehyde,
included in paraffin and 3 μm sections were cut with a microtome. Sections were
de-paraffinized using xylene, and rehydrated in a serial dilution of absolute
alcohol. Cells were permeabilized when necessary for intracellular staining
(PermWash 0.2% Triton X-100 in PBS) and stained using the antibodies listed in
acquired using Confocals PerkinElmer UltraVIEW ERS microscope (PerkinElmer Life
Sciences, Waltham, MA, USA) and deconvolved with Huygens Professional version
17.04 (Scientific Volume Imaging), using the Classic Maximum Likelihood
Estimation (CMLE) algorithm, with signal-to-noise ratio (SNR): 100
iterations.
6
Quantifying Keratin10, p53, and MCM in Cells
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2.4x104 cells were seeded in each well of a 4-well culture slide (Falcon) and cultured for 3 and 7 days. The cells were washed in PBS and fixed in 4% paraformaldehyde (PFA) in PBS for 30 min. at room temperature. The cells were permeabilised in PBS with 0.1% Triton X-100 (Promega) for 30 min., then washed in PBS. The cells were blocked in 10% normal goat serum (Cell Signaling Technology) in PBS for 1 hour. The antibodies used were anti-Keratin10 antibody (dilution 1:200, Thermo Fisher Scientific), anti-p53 (DO-1) antibody (dilution 1:300, Santa Cruz), anti-MCM antibody (dilution 1:100, Abcam) an anti-mouse Alexa 594-conjugated antibody (dilution 1:150, Thermo Fisher Scientific). For p53 and MCM, the signal was amplified using a Tyramide Signal Amplification Kit (Perkin-Elmer), according to the manufacturer's instructions. Finally, the cells were mounted in mounting medium (Agar Scientific) for visualization. The number of cells with positive signal were quantified using ilastik software.
7
Immunofluorescence and Oxidative Stress Assays
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Cells were cultured in 4-well culture slides (BD Biosciences, Franklin Lakes, NJ, USA), washed once with phosphate buffered saline (PBS), and then treated for overnight at 37 °C. Cells were then treated with 0.1% Triton X-100 to permeabilize the cells and blocked in 1% goat serum. HO-1 antibody was incubated overnight at 4 °C. The secondary antibody goat anti-rabbit IgG (H + L) (Invitrogen #A11034, Alexa Flour 488) was added for 1 h and mounted with DAPI-containing mounting media (Vectashield #H-1500). For ROS experiments, dihydroethidium (DHE), and dichloro-dihydro-fluorescein diacetate (DCFH-DA) reagents were added for 30 min at 37 °C. Slides were analyzed using Zeiss LSM 710.
8
Cell Viability Assay of DACC-Coated Dressing
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Cell viability after contact with either the DACC-coated dressing or the uncoated reference was determined [21 (link)]. In brief, cells were harvested through trypsin-EDTA (Gibco) treatment, seeded into 4-well culture slides (BD Biosciences) at a density of 40,000 cells/cm2, and cultured for 48 h to confluence. Stress controls and material samples were placed directly onto the cell layers. DMEM alone served as untreated (medium) controls. Cells were incubated for 1, 8, 24, and 48 h. Subsequently, the number of viable, active cells was determined using the photometric MTT assay (MTT Cell Proliferation Assay Kit, Invitrogen) according to the manufacturer’s recommendations. The absorbance was measured at 580 nm using a microplate photometer (POLARstar Galaxy, BMG Labtech). The number of viable cells was calculated as percentage of the medium control.
9
Mitochondrial Membrane Changes in MDA-MB-231 and MX-7 Cells
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For microscopic analysis of the mitochondrial membrane changes in MDA-MB-231 or MX-7 cells, cells were seeded (1 × 104) onto 4-well culture slides (BD Falcon, Bedford, MA), treated with recombinant VVs for 36 h, and then 5 μg/mL JC-1 were added for 20 min. Stained (nonfixed) cells were visualized with a fluorescence microscope, Axioskop 2 PLUS (Carl Zeiss, GmbH).
10
Cellular Uptake of Lipiodol Nanoemulsions
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To probe the cellular uptake of lipiodol nanoemulsions, 1.64 wt-% of Nile red was incorporated in the core of the nanoemulsions. HeLa cells were seeded in a chamber slide (4-well Culture Slides, BD Falcon, MA) at a density of 105 cells per well, and pre-incubated in DMEM at 37 °C for 24 h. The medium was then replaced by Nile red-loaded nanoemulsions at a concentration of 1.2 μg mL−1 in a fresh medium, and the cells were treated for 4 h at 37 °C. Afterwards, the nanoemulsions were removed, and the cells were washed several times with PBS. Then the cells were fixed using 4 wt-% formaldehyde for 30 min at room temperature. The nuclei of cells were stained using DAPI (1.5 mg mL−1) for 2 min, and the specimens were examined using a confocal laser scanning microscope (LSM510, Carl Zeiss, Oberkochen, Germany). Confocal images were quantitatively analyzed using ImageJ (U. S. National Institutes of Health, Bethesda, Maryland, USA).
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