The 3D tumorsphere cultures for both single and multisphere growth as well as tumorsphere invasion were performed as previously described [30 (link),31 (link)]. Briefly, for 3D single-spheroid formation, E0771 cells or their derivatives were seeded in a 96-well ultra-low attachment (ULA) plate at a density of 1.5 × 103 cells per well and centrifuged for 10 min at 125× g at room temperature. The plate was then incubated at 37 °C and imaged at day 3 and day 7 with a Leica DMi1 microscope to monitor the 3D single spheroid formation. For 3D spheroid invasion, E0771 cells or their derivatives were seeded in a 96-well ULA plate at a density of 1.5 × 103 cells per well and centrifuged for 10 min at 125× g at room temperature. For assessing for the invasive potential of the spheroids, 3 days post-formation of tumorsphere, each well was supplemented with 90 µL Matrigel (1:1 in complete culture medium) on top. The plate was then incubated at 37 °C and imaged at day 3 and day 7 with a Leica DMi1 microscope to monitor the 3D spheroid invasion. For 3D multi-spheroid formation, a 6-well plate was coated with polyhema overnight to allow polymerization. The following day, cells were plated at a density of 2 × 103 on each precoated well and imaged every 72 h for 11 days to monitor multi-spheroid formation with a Leica DMi1 microscope.
Dmi1 microscope
Manufactured by Leica
Sourced in Germany, United Kingdom, United States, Italy
The Leica DMi1 is a digital inverted microscope designed for routine imaging and documentation of biological samples. It features a high-resolution digital camera and supports various observation techniques, including brightfield and phase contrast.
Automatically generated - may contain errors
Lab products found in correlation
Las ez software, by Leica (3 mentions)
Mc120 hd, by Leica (2 mentions)
Mc170 hd camera, by Leica (2 mentions)
Oil red o, by Merck Group (2 mentions)
Mc170 camera, by Leica (2 mentions)
Application suite v4.12 program, by Leica (2 mentions)
Matrigel, by BD (2 mentions)
Application suite version 3, by Leica (2 mentions)
Mc120 hd camera, by Leica (2 mentions)
Senescence β galactosidase staining kit, by Cell Signaling Technology (2 mentions)
Apoptag peroxidase in situ apoptosis detection kit, by Merck Group (2 mentions)
Jsh 23, by Merck Group (1 mentions)
Mk 2206, by Cayman Chemical (1 mentions)
Bez235, by Cayman Chemical (1 mentions)
Erlotinib, by LC Laboratories (1 mentions)
Senescence detection kit, by Abcam (1 mentions)
Halotag alexa fluor 488 ligand, by Promega (1 mentions)
Clone okt3, by Thermo Fisher Scientific (1 mentions)
Rhil 2, by Merck Group (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
78 protocols using dmi1 microscope
1
3D Tumorsphere Cultures for Single, Multi, and Invasion Assays
2
Chemotaxis Assay for Macrophage Migration
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Migration assays were performed as previously described54 . Freshly isolated BMDMs (100,000 cells) were seeded in the top chamber of a 24-well PET membrane (8 mm pore size). Cells translocated to the lower chamber in response to exposure in the lower chamber of vehicle or 100 ng/ml CX3CL1 (472-FF; R&D) in the presence or absence of either an AKT inhibitor (MK-2206; 10 μM, Cayman), PI3K inhibitor (BEZ235; 10 μM, Cayman), EGFR inhibitor (erlotinib, 10 μM, LC laboratories) or NF-κB inhibitor (JSH23, 30 μM, Sigma-Aldrich) for 3 h. Cells in the upper chamber were removed with a cotton swab, and the filters were fixed with 70% ethanol and stained with 2% crystal violet. Filters were photographed on a Leica DMi1 microscope and total cell number was counted.
3
Antimigratory Evaluation of Bimetallic and Monometallic Compounds
4
Cytotoxicity Evaluation of ACF, MOFs, and Composites
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A human fibroblast (ATCC:
CRL-2522) and a rat H9C2(2-1) cardiomyoblasts (ATCC:CRL-1446; RRID:CVCL_0286)
were used to evaluate the cytotoxicity of ACF as well as MOFs and
ACF@MOF composites. The BJ cells were cultured in EMEM supplemented
with 10% of fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin
(100 μg/mL). The H9C2 myoblast was maintained in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with FBS (10%),
penicillin (100 U/mL), and streptomycin (100 μg/mL). Both cell
lines were incubated at 37 °C in a humidified atmosphere of 5%
CO2. Every 3 days, the cells were fed and subcultured to
prevent cell differentiation.
For the experiments, cells between
passages 7 and 20 were used. First, the cells were seeded (5 ×
105 cells/mL) on 96-well plates and incubated with ACF
for a period of 24 h in a concentration ranging from 0–50 μg/mL
(0 μM to 200 mM). Then, cell viability was evaluated using the
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT).
IC50 for ACF was determined.
Having established the toxic range
of ACF for the BJ and H9C2 cell
lines, a similar MTT test was performed for MOFs and ACF@MOF composites.
The images were taken with the use of a Leica DMi1 microscope.
CRL-2522) and a rat H9C2(2-1) cardiomyoblasts (ATCC:CRL-1446; RRID:CVCL_0286)
were used to evaluate the cytotoxicity of ACF as well as MOFs and
ACF@MOF composites. The BJ cells were cultured in EMEM supplemented
with 10% of fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin
(100 μg/mL). The H9C2 myoblast was maintained in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with FBS (10%),
penicillin (100 U/mL), and streptomycin (100 μg/mL). Both cell
lines were incubated at 37 °C in a humidified atmosphere of 5%
CO2. Every 3 days, the cells were fed and subcultured to
prevent cell differentiation.
For the experiments, cells between
passages 7 and 20 were used. First, the cells were seeded (5 ×
105 cells/mL) on 96-well plates and incubated with ACF
for a period of 24 h in a concentration ranging from 0–50 μg/mL
(0 μM to 200 mM). Then, cell viability was evaluated using the
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT).
IC50 for ACF was determined.
Having established the toxic range
of ACF for the BJ and H9C2 cell
lines, a similar MTT test was performed for MOFs and ACF@MOF composites.
The images were taken with the use of a Leica DMi1 microscope.
5
CAOV3 and SKOV3 Cell Morphology
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After culturing CAOV3 and SKOV3 cells with 6-ME for 24 h, cell morphologies were viewed under a LEICA DMI1 microscope and photographed using a LAS V4.12 digital camera (LEICA Corporation) with a ×10 eyepieces and ×20 objective.
6
Antiangiogenic Potential of Metal Complexes
7
Wound Closure Assay with Fibroblasts
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Post electroporation confluent healthy dermal fibroblasts were in 24 well plates at 100,000 cells/well, then left to adhere in standard DMEM media as described. After 24 h they were scratched with a p5 sterile pipette tip and images taken using a Leica DMi1 microscope every 8 h, analysis of this images conducted via Image J software and% wound closure obtained by deriving from the wound area.
8
Microscopic Imaging of Human NB Cells
9
Senescence Quantification in Cells
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Senescence levels were measured using the Senescence detection kit (Abcam, Cambridge, UK) according to manufacturer’s instructions. Cells were observed under a DMi1 microscope (Leica, Wetzlar, Germany) at 200× total magnification.
10
BMPR2 Localization in HMEC-1 Cells
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HMEC‐1‐HaloTag‐BMPR2 cells were treated overnight with BafA1 (20 nm ). Next, they were incubated in MCDB131 media containing the non‐permeable HaloTag Alexa Fluor 488 ligand (G1001; Promega) at a dilution of 1:10 × 103 for 15 min at 37°C. Cells were then washed three times with medium and images were recorded on a Leica DMi1 microscope.
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