All live-cell imaging was conducted using HeLa cells obtained from the University of California, Berkeley Cell Culture Facility. Cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium) F12 medium supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (PenStrep) and grown at 37°C and 5% CO2. Twenty-four hours before live-cell imaging, cells were plated onto eight-well chambered cover glass (Cellvis C8-1.5H-N) and incubated with 100 nM JF635 dye. Before imaging, cells were transferred to imaging medium consisting of DMEM F12–phenol red supplemented with 10% FBS and PenStrep. Cells were allowed to recover for at least 10 min in the incubator before movie acquisition.
C8 1.5h n
Manufactured by Cellvis
Sourced in United States
The C8-1.5H-N is a lab equipment product designed for a specific function. It provides a core feature without interpretation of its intended use. A detailed and unbiased description cannot be provided while maintaining conciseness.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Lsm800 confocal microscope, by Zeiss (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Axiovert microscope, by Zeiss (2 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
Lsm 800 microscope, by Zeiss (1 mentions)
Vybrant did, by Thermo Fisher Scientific (1 mentions)
P36961, by Thermo Fisher Scientific (1 mentions)
Ix83p2zf, by Olympus (1 mentions)
Actingreen 488 readyprobes reagent, by Thermo Fisher Scientific (1 mentions)
Lionheart fx automated microscope, by Agilent Technologies (1 mentions)
Lsm 880 microscope, by Zeiss (1 mentions)
Fluoromount g mounting medium, by Thermo Fisher Scientific (1 mentions)
Celltracker green cmfda dye, by Thermo Fisher Scientific (1 mentions)
Plan apochromat 63 1.4 oil immersion objective, by Zeiss (1 mentions)
Stage top incubator, by Tokai Hit (1 mentions)
Fugene 6 transfection reagent, by Promega (1 mentions)
Multiskan sky microplate reader, by Thermo Fisher Scientific (1 mentions)
Live dead cell imaging kit 488 570, by Thermo Fisher Scientific (1 mentions)
27 protocols using c8 1.5h n
1
Live-cell imaging of HeLa cells
2
HaloTag Labeling of HBEC Cells
3
FRAP Analysis of IRS-1 Droplets
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FRAP experiments were performed on a Zeiss LSM 800 microscope with a 63× oil immersion objective. C2C12 cells were seeded onto 8-well chamber slides (Cellvis, C8-1.5H-N). Cells were transfected with GFP-IRS-1 plasmids. After 24 h incubation, GFP-IRS-1 droplets were photobleached using a laser intensity of 80% at 480 nm (for GFP) and recovery was recorded for the indicated time. iFluorTM 488-FLAG-mIRS-1 droplets were photobleached using a laser intensity of 100% at 480 nm. The prebleached fluorescence intensity was normalized to 1 and the signal after bleaching was normalized to the pre-bleached level.
4
Visualizing EV Uptake in Macrophages
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Purified EVs were stained using Vybrant™ DiD (V22887; Thermo Fisher Scientific) lipophilic dye and washed twice in PBS using ultracentrifugation. PBS with 10% exosome-depleted FBS (A2720801; Thermo Fisher Scientific) was treated and “stained” in parallel as a negative control. Macrophages were differentiated and cultured on glass coverslips (C8–1.5H-N; Cellvis). Cells were dosed with 30 μL of “stained” controls or stained EVs (1 × 107 particles). Macrophages were then washed twice with PBS, fixed in 4% paraformaldehyde for 10 min, and stained with ActinGreen™488 ReadyProbes™ reagent (R37110; Thermo Fisher Scientific), and then with DAPI (EN62248; Thermo Fisher Scientific) before preservation in antifade mounting media (P36961; Thermo Fisher Scientific). Wells were then imaged using a spinning disc confocal microscope (IX83 P2ZF using cellSens Dimension v4.1, Olympus).30 (link)
5
Quantitative Cellular Uptake of Fluorescent PLGA Nanoparticles
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Both J774A.1 and MCF-7 cells were
seeded at the bottom of 8-well glass-bottomed chambers (#C8-1.5H-N,
Cellvis, Mountain View, CA) at a density of 50 000 cells/well
and left to adhere overnight. Next, at the desired time points, the
medium was replaced with DMEM containing the NR-labeled PLGA NPs at
17 μg/mL. The cells were washed using PBS and then fixed (prechilled
4% paraformaldehyde in PBS, 10 min), followed by counterstaining with
4′,6-diamidino-2-phenylindole (DAPI, 300 nm in PBS,
10 min), and 3 washing steps (PBS). The cells were immersed in a Fluoromount-G
mounting medium (Thermo Fisher Scientific). Visualization of monocytes-macrophages
was carried out using a Zeiss LSM 880 microscope (Zeiss, Oberkochen,
Germany) with a 63× oil objective (Objective Plan-Apochromat
63×/1.4 Oil DIC M27). Signal detection (pinholes 80 μm)
was observed at channels Ch 1 (excitation 405 nm, emission 415–473
nm, DAPI) and Ch 2 (excitation 552 nm, emission 579–636 nm).
Similarly, NPs’ cellular uptake by MCF-7 cells was analyzed
using a BioTek Lionheart FX Automated Microscope for both DAPI and
Rh-B (excitation 560 nm, emission 583 nm) field.
seeded at the bottom of 8-well glass-bottomed chambers (#C8-1.5H-N,
Cellvis, Mountain View, CA) at a density of 50 000 cells/well
and left to adhere overnight. Next, at the desired time points, the
medium was replaced with DMEM containing the NR-labeled PLGA NPs at
17 μg/mL. The cells were washed using PBS and then fixed (prechilled
4% paraformaldehyde in PBS, 10 min), followed by counterstaining with
4′,6-diamidino-2-phenylindole (DAPI, 300 n
10 min), and 3 washing steps (PBS). The cells were immersed in a Fluoromount-G
mounting medium (Thermo Fisher Scientific). Visualization of monocytes-macrophages
was carried out using a Zeiss LSM 880 microscope (Zeiss, Oberkochen,
Germany) with a 63× oil objective (Objective Plan-Apochromat
63×/1.4 Oil DIC M27). Signal detection (pinholes 80 μm)
was observed at channels Ch 1 (excitation 405 nm, emission 415–473
nm, DAPI) and Ch 2 (excitation 552 nm, emission 579–636 nm).
Similarly, NPs’ cellular uptake by MCF-7 cells was analyzed
using a BioTek Lionheart FX Automated Microscope for both DAPI and
Rh-B (excitation 560 nm, emission 583 nm) field.
6
Live-Cell Imaging of Microglial Phagocytosis
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For live-cell imaging, 50,000 iPSC-microglia or THP-1 macrophages were cultured in a vitronectin-coated eight-chambered cover glass dish (Cellvis, C8-1.5H-N). THP-1 macrophages were labeled with 2 µM CellTracker Green CMFDA dye (Invitrogen, C2925) in HBSS containing Ca++ and Mg++ (Gibco, 14025-092) for 30 min at 37 °C. Medium was replaced with cell medium containing 2 µg/ml β-Amyloid1-42-HiLyte Fluor 555 and cells were imaged 1 h following treatment.
7
FRAP Analysis of Protein Dynamics
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The 293T cells were seeded into eight-well chambered cover glass (Cellvis, #C8-1.5H-N) and labeled after 24 h with 10 µg/ml Alexa Fluor™ 647-conjugated antibody in FluoroBrite for 10 min at 37 °C. FRAP experiments were performed on a confocal microscope (Zeiss LSM700) using the Plan-Apochromat 63 × 1.4 oil-immersion objective and 2% 639 nm solid-state laser excitation intensity for image acquisition. Every 1.5 s an image was recorded for a total acquisition time of 120 s and after three initial images a bleaching step was performed using 100% laser intensity of the 639 nm and 555 nm laser with a pixel dwell time of 37.6 µs. Frame size, bleaching area, as well as laser intensities and imaging speed was kept constant for all FRAP experiments. All experiments were performed at 37 °C and 5% CO2 using a stage top incubator (Tokai Hit). FRAP data were analyzed with the software Zen system 2012 and custom-made python code.
8
Transfection of HEK293 cells with TDiP aggregates
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HEK293 cells were seeded to 8-chamber dishes (Cellvis #C8-1.5H-N) such that they reached 50% confluence 16 hours post-seeding. Cells were transfected with a control mCherry expression vector made in-house (pcDNA3.1-P2A-mCherry-T2A) using Fugene 6 Transfection reagent according to manufacturer protocol. 24 hours later, 10.0 μg (A280) of TDiP aggregates per well (∼80 mm2), were transfected into cells using ProteoJuice Transfection reagent (Millipore Sigma #71281). Ratios were adapted for chamber dish from manufacturer protocol and De Rossi et al., 2021. 4 hours following transfection, cells were returned to complete media and imaged on a 37°C heated stage in a humidified, 5% CO2 chamber.
9
Protein Phase Separation Assay
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The purified proteins were added to phase separation Buffer (50 mM Tris-HCl pH 7.5, 10% glycerol, 1 mM DTT, 2% PEG-8000 (Sangon biotech, A100159)). The concentration of NaCl was adjusted to the indicated concentrations. The protein solution was loaded onto an 8-well chamber (Cellvis, C8-1.5H-N) for 5 min at room temperature and then imaged using a Zeiss LSM 800 confocal microscope with a 63× objective (Carl Zeiss). For temperature-mediated phase separation, the 8-well chamber was first incubated in 37 °C for 5 min and then shifted to 4 °C for 5 min. For 1,6-Hexanediol (TCI, H0099)-mediated phase separation, 1,6-Hexanediol was present at the indicated concentrations. The concentrated proteins were treated with 0.1 mg/mL RNase A (Axygen, AP-MD-P), 1 U DNase (Thermo Scientific, EN0521) or 0.1 mg/mL BSA for 1 h at room temperature to examine the effects of RNA or DNA on LLPS. Droplet turbidity OD600 was measured by using a Thermo Multiskan Sky microplate reader.
10
Quantifying Live and Dead Neuronal Cells
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To determine the proportion of live and dead cells, we plated ~ 40,000 iNs on Matrigel coated 8-chamber glass slide (Cellvis C8–1.5H-N) and cultured them for 11 days post-differentiation after which incubation with Apilimod and PFF were for 10 days as described elsewhere. Briefly, cells in 50 μL imaging media (phenol red-free Neurobasal medium supplemented with 1% B-27, 10 ng/mL BDNF, 10 ng/mL CNTF, and 10 ng/mL GDNF) were incubated for 15 minutes at room temperature with 50 μL 2x stock solution of LIVE/DEAD Cell Imaging Kit 488/570 (Thermo Scientific R37601) containing live cell Calcein AM and dead cell stain BOBO-3-Iodide, followed by spinning disc confocal imaging with 40x magnification and counting.
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