96-well microplates (CLS3603; Sigma-Aldrich) were coated with 50 µg/ml type I collagen (354236; Thermo Fisher Scientific) or type IV collagen (356233; Thermo Fisher Scientific) at 37°C for 30 min. After washing with PBS, wells were blocked by incubation with 0.5 mg/ml heat-treated BSA in PBS-ABC (containing 1 mM CaCl2 and 1 mM MgCl2) for 1 h at 37°C (Levy et al., 2000 (link)). Freshly isolated keratinocytes (2 × 104) were plated in each well in mKER medium and incubated at 37°C for 2 h. The nonadherent cells were washed off with PBS-ABC. The adherent cells were lysed with medium containing 1% Triton X-100. The relative efficiency of cells to matrix was determined by measuring the level of LDH, which is released upon cell lysis, using the using the Cytotoxicity Detection Kit (4744926001; Sigma-Aldrich).
Cls3603
Manufactured by Merck Group
The CLS3603 is a laboratory equipment product manufactured by Merck Group. It is a compact and versatile piece of equipment designed for general laboratory use. The CLS3603 provides core functionalities for common laboratory tasks, but a detailed description of its intended use is not available.
Automatically generated - may contain errors
Lab products found in correlation
Synergy h1 microplate reader, by Agilent Technologies (2 mentions)
Type 4 collagen, by Thermo Fisher Scientific (1 mentions)
Collagen type 1, by Thermo Fisher Scientific (1 mentions)
Cytotoxicity detection kit, by Merck Group (1 mentions)
Celltiter glo luminescent cell viability assay, by Promega (1 mentions)
Spectramax i3x spectrophotometer, by Molecular Devices (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
D5671, by Merck Group (1 mentions)
Fugene hd, by Promega (1 mentions)
Fluostar omega microplate reader, by BMG Labtech (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
9 protocols using cls3603
1
Keratinocyte Adhesion to Collagen Matrices
2
hESC Viability Assay in 96-well Plates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
hESCs were passaged as described above (H1-hESC cell culture and CRISPR), and cells were counted to ensure even plating across genotypes. Cells were plated in Geltrex-coated black-sided clear-bottomed 96-well tissue culture dishes (Sigma-Aldrich, CLS3603). Wells on the edge of the dish were excluded. Cell viability was measured using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, G9242) following kit instructions. Significance was measured using Student’s t tests.
3
Automated GFP Induction Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
96 well plate with lid (Sigma #CLS3603) contained 300 μL per well and scanned using a Biotek Synergy H1 Microplate Reader with the following settings: incubate at 37°C with constant, maximum shaking (orbital frequency: 807 cpm). Every 10 min, read A600, and GFP fluorescence (485 nm excitation, 515 nm emission) for a total of 8 h. Additional plate settings: autoscale GFP, scale to low wells [Receiver + 50% negative sender wells (3 wells)], 200 scale value. All initial OD600 were 0.4 after dilution with sender supernatant. Negative sender sample consisted of 150 μL of negative sender cells resuspended in fresh LB/amp and 150 μL of filtered negative sender supernatant. GFP+ controls consisted of 150 μL GFP+ cells diluted in fresh LB/amp and 150 μL of filtered negative sender supernatant. Cells induced with 10% supernatant consisted of 150 μL of receiver cells diluted in fresh LB/amp, 30 μL filtered sender supernatant, 120 μL filtered negative sender supernatant. Cells induced with 25% supernatant consisted of 150 μL of receiver cells in fresh diluted LB/amp, 75 μL filtered sender supernatant, and 75 μL filtered negative sender supernatant. Cells induced with 50% supernatant consisted of 150 μL of receiver cells diluted in fresh LB/amp, and 150 μL filtered sender supernatant.
4
Quantifying Bacterial EPS Matrix Formation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EPS matrix detection was performed as described previously 72. In brief, 130 l diluted bacterial cultures (1:50 from an overnight bacterial) in LB, LB + 0.15% bile or LB + 0.2% NaGCH were grown in a black 96-well clear flat bottom polystyrene plate (Corning® Cat. No. CLS3603 Sigma) overnight at 37 °C. Planktonic bacteria was transferred to a new 96-well polystyrene microtiter plate (Corning®, Cat. No. CLS3595 Sigma). Wells in the black plate were fixated with formaldehyde/glutaraldehyde, rinsed with PBS and stained with 150 l of Concanavalin A (ConA) conjugated to fluorescein isothiocyanate (FITC) (25 g/ml) for 15 min. After rinsing with PBS, the amount of retained ConA-FITC was measured using a SpectraMax i3x spectrophotometer (Molecular Devices) at 480 nm. This assay was performed in four independent experiments.
5
Photoswitchable Enzyme Inhibitor Kinetics
6
Quantifying Intracellular Triglycerides using Nile Red
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A modified Nile Red staining method28 (link) was used to quantify intracellular TAGs. Briefly, cells were harvested by centrifugation (3000 g for 10 min at 4 °C), supernatant was removed and the pellet resuspended in fresh 0.5 M ATCC-1174 DA media to an OD680 of 0.3. Triolein was used as a standard for determining neutral lipid concentrations by Nile Red (Fig. S10 ). Two hundred microliters of triolein standards (40, 20, 10, 5, 2.5, 0 μg/mL) and cell suspensions were loaded as technical triplicates onto a 96-well black, clear bottom plate (CLS3603; Sigma-Aldrich). Prior to staining, Nile red stock is diluted in acetone to obtain a working solution (25 μg/mL), and 2 μL of the Nile red working solution is added to each well of sample and standard, followed by a 5 min incubation in the dark. Fluorescence of each sample was detected using a microplate reader (Infinite M200 PRO, Tecan) at excitation and emission wavelengths of 524 nm and 586 nm. Fluorescence imaging of Nile Red-stained cells was performed with an automated fluorescence microscope (Olympus BX63). Acquisition and processing of data was done using the cellSens software.
7
Nanobody-TagBFP Transfection in Mammalian Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All constructs used in mammalian cell transfection experiments were cloned into the pCAG plasmid. Plasmid DNAs encoding nanobody-TagBFP sequences were transfected into both 293T cells and HeLa cells. Experiments were first conducted in 293T cells, tested across three replicates, and later tested in HeLa cells. One day prior to plasmid transfection, cells were plated in black, clear bottomed 96-well plates (CLS3603, Sigma-Aldrich) at roughly 10,000 cells/well and incubated at 37°C, and 5% CO2. For transfection of cells in each well, 5 µl of serum-free media was added to 200 ng of plasmid DNA (100 ng CAG-nanobody-TagBFP plasmid and 100 ng CAG-dsRed plasmid [Addgene plasmid 11151], Matsuda and Cepko, 2004 (link) or CAG-H2B-mCherry plasmid). For target-binding experiments, an additional 100 ng of NLS target expressing plasmid or 100 ng of an off-target, control NLS expressing construct was added. 1 µl (or 1.5 µl for target-binding experiments) of 1 mg/ml PEI (Polysciences, 24765-2) dissolved in water was added to DNA in media and vortexed for 10 s. Resulting transfection mix was left to sit at room temperature for 10 min before being added to cells.
8
Measuring Mitochondrial Membrane Potential
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lines stably expressing Cytc variants were seeded at a density of 20,000 cells/well onto black 96-well plates (Costar, CLS3603, Sigma, Ronkonkoma, NY) and allowed to grow to 80–90% confluence. To assess relative changes in ΔΨm, cells were incubated for 1 h in phenol red- and FBS-free medium containing 1 µM JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolyl-carbocyanine iodide, Molecular Probes, Inc.). JC-1 can selectively enter mitochondria and exists as a monomer at low membrane potential or at low concentration, emitting green fluorescence. At higher membrane potential, JC-1 aggregates and emits red fluorescence. Fluorescence was measured in PBS using a Synergy H1 microplate reader (BioTek Instruments, Inc., Winooski, VT) with excitation: 485 nm; emission: 527 nm, and excitation: 485 nm; emission: 590 nm, respectively. Data were presented as the ratio of red to green fluorescence.
9
Optimized Luciferase Assay in HeLa Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HeLa cells (ATCC CCL-2) were cultured in 25-cm2 flasks at 37 °C, 5% CO2 in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, D5671) supplemented with 10% fetal calf serum, 2 mM l -glutamine, and penicillin/streptomycin. Cells were passaged and transferred to 96-well assay plates (Sigma-Aldrich, CLS3603) 36 h prior to transfection. The time between plating and transfection was optimized experimentally for maximal transfection efficiency. Transfection was carried out using FuGENE HD (Promega, E2311). Each transfection reaction was set up according to the manufacturer’s protocol in 100 µl using a total of 2 µg of plasmid DNA and 6 µl of FuGENE HD. Luciferase assays were performed 36 h after transfection with the Dual-Glo Luciferase Assay System (Promega, E2920). Firefly and Renilla luciferase expression was quantified by measuring luminescence with no emission filter on the FLUOstar Omega microplate reader (BMG LABTECH). For each assay, three biological replicates (two for psiCHECK-2:Ci-UTR without miR-2768) on separate plates were performed at separate times and relative Renilla and firefly luciferase activities were recorded. Each biological replicate consisted of three or five technical replicates. The mean Renilla:firefly ratio for pCS2+:2768 cotransfected with blank psiCHECK-2 was used to normalize readings across plates.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!