EC migration was determined using a transwell migration assay. Briefly, control and Wt1KO ECs were cultured in a serum-free EBM-2 medium overnight before the assay. Trypsinized cells were seeded on gelatin-coated transwell membranes with 8 µm pores (Corning, 351152) at a density of 40,000 cells per well and allowed to migrate toward EGM-2 medium containing 2% FBS over a 16 h. After cell migration, solutions in the basal chamber were removed and basal membranes were washed twice with PBS. Next, 900 µl of 5 µM calcein solution (Invitrogen, C3100MP) dissolved in EBM-2 were added to the basal chamber and left to incubate at 37°C for 30 min. Calcein solution was washed and transwells were transferred to a new 24-well plate, maintaining the seeding solution in the bottom chamber until calcein fluorescence was read by measuring the 480/530nm emission on an Infinite 200 plate reader.
C3100mp
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The C3100MP is a laboratory centrifuge designed for general-purpose applications. It features a compact and durable construction, with a maximum speed of 3,100 rpm and a maximum relative centrifugal force (RCF) of 1,762 x g. The centrifuge accommodates a variety of rotor types and sample volumes, making it suitable for a wide range of sample preparation and separation needs.
Automatically generated - may contain errors
Lab products found in correlation
P3566, by Thermo Fisher Scientific (5 mentions)
E1169, by Thermo Fisher Scientific (4 mentions)
Calcein am, by Thermo Fisher Scientific (3 mentions)
P1304mp, by Thermo Fisher Scientific (3 mentions)
Ix81 confocal microscope, by Olympus (2 mentions)
P50g 1.5 39 f, by MatTek (2 mentions)
Fluoview 1000, by Olympus (2 mentions)
Sp8 3 sted super resolution confocal microscope, by Leica (2 mentions)
Bz x700 microscope, by Keyence (2 mentions)
Axio observer z1 inverted microscope, by Zeiss (2 mentions)
Axioplan 2 microscope, by Zeiss (1 mentions)
Bay 876, by Cayman Chemical (1 mentions)
Wzb 117, by Cayman Chemical (1 mentions)
Fcs express 7, by BD (1 mentions)
Accuri c6, by BD (1 mentions)
Spectramax plus 384 microplate reader, by Molecular Devices (1 mentions)
Inverted microscope, by Nikon (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Matrigel, by Corning (1 mentions)
55 protocols using c3100mp
1
Transwell Assay for EC Migration
2
Choroid Plexus Epithelial Cell Viability Assay
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Calcein acetoxymethyl (calcein-AM) staining was employed to verify choroid plexus epithelial cell viability [33 (link)]. Viable cells contain active intracellular esterases that cleave the AM group from the calcein, resulting in a bright fluorescent signal from these cells. Choroid plexus was incubated in aCSF containing calcein-AM (16.7 µM, C3100MP, Invitrogen) for 10 min at room temperature and transferred to microscope slides for microscopy. Images were acquired using Zeiss Axioplan 2 microscope equipped with epifluorescence and interference filters with a 702 moni AxioCam and using Zeiss Zen Black software.
3
Glucose Transporter Inhibition in Hypoxic Rat Nucleus Pulposus Cells
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Rat NP cells were isolated as reported previously by Risbud and colleagues (5 (link)). Rat NP cells were maintained in DMEM supplemented with 10% FBS and antibiotics. Cells were cultured in a Hypoxia Work Station (Invivo2 400) with a mixture of 1% O2, 5% CO2, and 94% N2. To investigate the effect of GLUT inhibition, NP cells were treated with 1) a cocktail of GLUT1 inhibitors (BAY-876, 0.01, 0.1 μM, Cayman, 19961; or WZB-117, 1, 10 μM, Cayman, 19900) for 1 to 72 hours or 2) a GLUT1, 2, and 3 inhibitor (Glutor, 0.05, 0.1, and 0.25 μM; Sigma, SML2765) for 6 to 72 hours. Viability measurements following treatment of NP cells in hypoxia with BAY-876, WZB-117, and Glutor for 24–72 hours were performed using a Calcein AM cell viability assay as per manufacturer’s instructions (Invitrogen, C3100MP). All in vitro experiments were performed in hypoxia (1% O2) at least 3–6 independent times with 4 replicates/experiment/group and data represented as mean ± SEM.
4
Analyzing Nanoparticle Uptake in Primary Human Aortic Endothelial Cells
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Primary Human Aortic Endothelial
Cells (HAEC) were purchased from Lifeline Cell Technologies (FC-0014)
and cultured in Vasculife VEGF cell culture medium containing 2% FBS,
10 mMl -glutamine, 0.75 U/ml heparin sulfate, 1.0 μg/mL
hydrocortisone hemisuccinate, 50 μg/mL ascorbic acid, 15 ng/mL
rh IGF-1, 5 ng/mL rh FGF Basic, and 5 ng/mL rh EGF at 37 °C in
a humidified 5% CO2 incubator. Calcein and propidium iodide
assays (Invitrogen Part #C3100MP and P3566, respectively) were performed
on the cells. P5 cells were plated on collagen-coated 50 mm glass
bottom culture dishes (MatTek Part # P50G-1.5-39-F), grown to ∼95%
confluency, and then incubated for 20 h with NaGdF4:Eu
or NaGdF4:Eu@Au nanoparticles suspended in VEGF cell culture
medium at 50 μg/mL and 250 μg/mL. The culture medium was
then gently aspirated out, replaced with 3 μM Calcein AM in
DPBS and incubated for 15 min, the last 5 of which propidium iodide
nucleic acid stain was added to a final concentration of 4 μM.
The culture dishes were placed on an inverted Olympus IX81 Confocal
Microscope and imaged using Fluoview 1000. Image J analysis was performed
on the images by setting a threshold to convert them to a black and
white format. Particle analysis was used to count the number of apparent
particles and the total surface area covered by particles. Particles
smaller than 200 × 200 pixels were excluded.
Cells (HAEC) were purchased from Lifeline Cell Technologies (FC-0014)
and cultured in Vasculife VEGF cell culture medium containing 2% FBS,
10 mM
hydrocortisone hemisuccinate, 50 μg/mL ascorbic acid, 15 ng/mL
rh IGF-1, 5 ng/mL rh FGF Basic, and 5 ng/mL rh EGF at 37 °C in
a humidified 5% CO2 incubator. Calcein and propidium iodide
assays (Invitrogen Part #C3100MP and P3566, respectively) were performed
on the cells. P5 cells were plated on collagen-coated 50 mm glass
bottom culture dishes (MatTek Part # P50G-1.5-39-F), grown to ∼95%
confluency, and then incubated for 20 h with NaGdF4:Eu
or NaGdF4:Eu@Au nanoparticles suspended in VEGF cell culture
medium at 50 μg/mL and 250 μg/mL. The culture medium was
then gently aspirated out, replaced with 3 μM Calcein AM in
DPBS and incubated for 15 min, the last 5 of which propidium iodide
nucleic acid stain was added to a final concentration of 4 μM.
The culture dishes were placed on an inverted Olympus IX81 Confocal
Microscope and imaged using Fluoview 1000. Image J analysis was performed
on the images by setting a threshold to convert them to a black and
white format. Particle analysis was used to count the number of apparent
particles and the total surface area covered by particles. Particles
smaller than 200 × 200 pixels were excluded.
5
Quantifying Cell Adhesion via Calcein-AM
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Adapted from Weetall et al (25 (link)). V-bottom 96-well plates were seeded with 2 x 104 cells/well and left in 37°C overnight. Calcein-AM-labeled cells (2uM Calcein-DMSO solution, Invitrogen #C3100MP) were subsequently seeded at 1.5 x 104 cells/well to the plate (negative control: wells with overnight-seeded cells but no Calcein-labeled cells; positive control: empty wells with Calcein-labeled cells added). Plates were incubated at 37°C for 2 hours, then centrifuged at 75 g for 10 minutes. Nonadherent cells accumulated at the bottom of the wells and fluorescence at the bottom of the well was quantified. Log fold change in fluorescence between test wells and positive control wells was recorded. Assay was repeated with media containing 100 IU/ml IFN-α; overnight-seeded cells were treated with IFN-α for 48 hours prior to seeding in v-bottom plates, then were seeded in media with IFN-α for 24 hours, while Calcein-labeled cells were treated with IFN-α for 72 hours prior to Calcein labeling, seeding, and incubation in v-bottom plate for 2 hours (they were also seeded in media containing IFN-α).
6
Calcein Green Staining of VERO E6 Cells
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Uninfected VERO E6 cells were subjected to the full inactivation protocol (S1). Cells were then stained with 10uM of calcein green (Invitrogen, C3100MP) and incubated for 20 minutes at room temperature. Flow cytometry data were then collected and analyzed using instrument BD Accuri C6 and FCSexpress 7 software (Version 7.12.0007, respectively).
7
Mitochondrial Function and ROS Measurement
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For measuring mPTP opening, cells were washed twice with Hank’s buffer, and then stained with 1 µg/µL calcein (C3100MP, Invitrogen) in the presence of 1 µM CoCl2 for 25 min at 37 °C, and washed twice with Hank’s buffer before imaging. For measuring ΔΨm, cells were stained with 50 nM Dilc(5) (M34151, Molecular Probes) for 25 min in 37 °C, and then washed twice with serum-free medium before imaging. For measuring ROS levels, cells were incubated with 1 µM carboxy-DCFH-DA (DCFH-DA) (S0033, Beyotime) in serum-free medium for 25 min at 37 °C. The fluorescence was detected by Zeiss confocal microscope. The fluorescence intensities were analyzed as in our previous report26 (link).
8
Cell Adhesion Assay for Endothelial Cells
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Cell adhesion assay was performed as previously described in Ref. [25 ]. Briefly, HUVECs were plated on a 96‐well fluorescence plate and treated as described in the cell culture methods part. THP‐1 cells were washed with serum‐free RPMI 1640 medium and suspended at 5 × 106 cell/mL in serum‐free medium with 5 µmol/L of Calcein AM (C3100MP; Invitrogen). Cells were incubated for 30 minutes and the labelling reaction was stopped by adding the same volume of cell growth medium. Then cells were washed with growth medium twice and resuspended in growth medium at 5 × 105 cell/mL. After 16 hours of the relevant treatment, HUVECs were washed once with THP‐1 cell growth medium, and 200 µL Calcein AM‐labelled THP‐1 cells were added to each well. After 1 hour of incubation, the non‐adherent cells were removed carefully and adherent cells were washed with RPIM 1640 medium five times. The images were captured by a microscopy (Carl Zeiss). The mean fluorescence intensity per view was used to show the numbers of THP‐1 cells attached to ECs and quantified from randomly acquired images.
9
Evaluating Cell Viability via Calcein Assay
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Cell viability was monitored by the calcein assay, which examined cells stained with calcein acetoxymethylester (AM) (C3100MP, Invitrogen, USA). Cultured ORNs were treated with a 10 μM odorant mixture for 6 h and 18 h, respectively, prior to incubation with calcein AM solution (final concentration 3 μM) for 30 min. Cell viability was measured at wavelengths of 485 nm (excitation) and 535 nm (emission) using a microplate reader (SpectraMax Plus 384 Microplate Reader, Molecular devices, USA).
10
Transwell Migration Assay with Exosomes
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Transwell chambers (6.5 mm) (Corning Costar, Cambridge, MA, USA) with 8.0-µm pore polycarbonate membranes were coated with Matrigel. HUVECs (20,000 cells/well) were incubated in the upper chamber at 37 °C in 5% CO2 and allowed to migrate for 8 h toward the lower chamber. Some HUVECs were co-cultured with exosomes for 72 h. The number of cells that migrated through the membrane to the lower chamber was measured after 8 h with calcein-AM (Invitrogen, C3100MP; 50 µg).Cells in the lower chamber were counted in three random microscopic fields using an inverted microscope (Nikon, Japan).
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