Cyquant cell lysis buffer

Manufactured by Thermo Fisher Scientific

Sourced in United States

The CyQUANT Cell Lysis Buffer is a solution designed to facilitate the lysis of cells for subsequent analysis. It provides a consistent and efficient method for releasing cellular contents, including DNA, RNA, and proteins, from a variety of cell types.

Automatically generated - may contain errors

Lab products found in correlation

Spelling variants of this product

Lysis buffer (523 citations) Cell lysis buffer (171 citations) CyQUANT (133 citations)

7 protocols using cyquant cell lysis buffer

Cytokine Secretion Quantification in Macrophages

Check if the same lab product or an alternative is used in the 5 most similar protocols

The secretions of TNF-α and IL-1β were measured in the conditioned media from the cultured MΦs using commercially available, enzyme-linked immunosorbent assay kits (Novex^®/Life Technologies), according to the instructions from the manufacturer. All samples were measured at 450 nm and referenced at 630 nm using a microplate reader (Instruments ELX800, BioTek). Intracellular proteins were collected by transferring samples to microcentrifuge tubes containing 1× CyQUANT cell lysis buffer (Life Technologies) and quantified by mixing the samples with a Pierce™ BCA Protein Assay Kit (Life Technologies). The values were read at 570 nm using the microplate reader. Serially diluted recombinant standards for each cytokine of interest were used to create a standard curve for determining cytokine concentrations in the conditioned media that were normalized to the amount of intracellular total protein (and expressed as pg/μg cellular protein).

Hsu Y.T., Wu C.Y., Guan Z.Y., Sun H.Y., Mei C., Chen W.C., Cheng N.C., Yu J, & Chen H.Y. (2019). Characterization of Mechanical Stability and Immunological Compatibility for Functionalized Modification Interfaces. Scientific Reports, 9, 7644.

+ Open protocol

+ Expand

MSC Leukocyte Binding Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

MSCs (2 × 10⁴ cells/well) were plated in 24-well plates (Corning, Corning, NY) in 2% CCM and allowed to adhere for at least 6 h and then activated as above. U937 cells were labeled with CellTracker Red CMPTX (Life Technologies) following manufacturer's protocol. Briefly, U937 cells (2 × 10⁶ cells/ml) were resuspended in pre-warmed CellTracker dye working solution (10 μM in serum–free RPMI) and incubated at 37°C. After 45 min, cells were resuspended in complete RPMI media. Following 30 min incubation at 37°C, cells were washed and resuspended at 10⁶ cells/ml in complete RPMI media. Fluorescent-labeled U937 cells (5 × 10⁵ cells/well) were allowed to bind to MSCs for 1 h at 4°C, as previously described16 (link), to determine the extent to which activated MSCs could bind leukocytes without any significant physiological changes to the cells upon contact. Next, the plate was washed with cold PBS for removal of unbound cells and remaining cells were lysed with CyQUANT® Cell lysis buffer (Life Technologies) and 100 μl removed for fluorescence measurement. The number of bound cells/well was calculated based on a standard curve generated with a serial dilution of known numbers of fluorescent-labeled U937.

Kota D.J., DiCarlo B., Hetz R.A., Smith P., Cox C.S, & Olson S.D. (2014). Differential MSC activation leads to distinct mononuclear leukocyte binding mechanisms. Scientific Reports, 4, 4565.

+ Open protocol

+ Expand

Measuring Mitochondrial Respiration in Adipocytes

Check if the same lab product or an alternative is used in the 5 most similar protocols

To directly assess the effects of MTIF3 on mitochondrial respiration in adipocytes we used the Seahorse XF (Seahorse Bioscience, North Billerica, MA) to measure cellular respiration OCR under different conditions. hWAs-iCas9 cells were seeded at 8000 cells/well in a Seahorse 24-well plate, then differentiated and induced for MTIF3 knockout or with scrambled control, as described above. Then, cells were adapted in 1 g/l growth medium (31885049, Thermo Fisher Scientific) for 3 days. Mitochondrial function was then assessed using the Seahorse XF-24 instrument according to a protocol optimized for the adipocyte cell line. Briefly, to measure OCR independent of oxidative phosphorylation, 2 μM oligomycin (O4876, Sigma-Aldrich) was added to the cells. Subsequently, 2 μM FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) (C2920, Sigma-Aldrich) and 5 μM respiratory chain inhibitors: rotenone (R8875, Sigma-Aldrich) and antimycin A (A8674, Sigma-Aldrich) were added to measure maximal respiration and basal rates of non-mitochondrial respiration. Cells were then frozen at −80°C for at least 4 hr, then the plate was dried, and DNA was extracted with CyQUANT Cell Lysis Buffer (C7027, Thermo Fisher Scientific). Total DNA was then quantified by Quant-iT PicoGreen dsDNA Assay Kit (P7589, Thermo Fisher Scientific) against a lambda DNA-generated standard curve.

Huang M., Coral D., Ardalani H., Spegel P., Saadat A., Claussnitzer M., Mulder H., Franks P.W, & Kalamajski S. (2023). Identification of a weight loss-associated causal eQTL in MTIF3 and the effects of MTIF3 deficiency on human adipocyte function. eLife, 12, e84168.

+ Open protocol

+ Expand

Dual-Luciferase Assay for miRNA Functional Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

A dual-luciferase assay was performed as described previously (24 (link)). Briefly, cells were seeded in a 24-well plate at ~80% confluence and transfected with miRNA expression vector, reporter vector and the pRL-TK control vector encoding Renilla luciferase (Biovector Science Lab, Inc., Beijing, China), using Invitrogen Lipofectamine 2000 according to the manufacturer's instructions. Following 24 h of transfection, cells were harvested, lysed by CyQuant^® cell Lysis Buffer (Thermo Fisher Scientific, Inc.) and analyzed using the Dual-Luciferase Reporter Assay System kit (Promega Corporation). All experiments were performed in triplicate.

Ma C., Zheng C., Bai E, & Yang K. (2016). miR-101 inhibits glioma cell invasion via the downregulation of COX-2. Oncology Letters, 12(4), 2538-2544.

+ Open protocol

+ Expand

Measuring Mitochondrial Respiration in Adipocytes

Check if the same lab product or an alternative is used in the 5 most similar protocols

To evaluate mitochondrial respiration, Seahorse XF (Seahorse Bioscience, USA) was used to measure oxygen consumption rate (OCR) in white adipocytes. Briefly, hWAs cells were seeded in a Seahorse 24-well plate and induced to differentiate using protocols described above. OCR was recorded continuously by sequentially adding 2 μmol/l oligomycin (EMD Chemicals, USA), 2 μmol/l FCCP and 5 μmol/l of the respiratory chain inhibitor rotenone at the indicated time points. After the measurement, the cell plate was then frozen at −80°C for at least 4 h, then the plate was dried and DNA was extracted with CyQUANT Cell Lysis Buffer (C7027, Thermo Fisher Scientific). Total DNA was then quantified using Quant-iT PicoGreen double-stranded DNA (dsDNA) assay kit (P7589, Thermo Fisher Scientific) against a lambda DNA-generated standard curve.

Huang M., Claussnitzer M., Saadat A., Coral D.E., Kalamajski S, & Franks P.W. (2023). Engineered allele substitution at PPARGC1A rs8192678 alters human white adipocyte differentiation, lipogenesis, and PGC-1α content and turnover. Diabetologia, 66(7), 1289-1305.

+ Open protocol

+ Expand

Cell Viability and Proliferation Assays

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cell behavior was assessed for all 2D and 3D cultures after 2 and 10 days of seeding or printing. Cellular metabolic activity was assessed using alamarBlue^® assay (Pierce Biotechnology, Rockford, IL, USA), with sample absorbance measured using a Biotek 800TS microplate reader (Winooski, VT, USA) at 570 and 600 nm. DNA concentration was determined using the Quant-iT PicoGreen^® assay (Molecular Probes, Invitrogen, Eugene, OR, USA) according to the manufacturer’s protocol with slight modification. Briefly, a series of freezing and thawing cycles, in combination with a Cyquant Cell Lysis Buffer (Invitrogen, Eugene, OR, USA), was used to lyse the cells for DNA quantification. Hydrogels were dissociated using a 1:1 ratio of 0.2M sodium citrate solution and 1X cell lysis buffer. Cell viability was also qualitatively assessed for 3D cell-laden hydrogels using a LIVE/DEAD^® Viability/Cytotoxicity assay (Molecular Probes, Invitrogen, Eugene, OR, USA) according to manufacturer specifications. Images were collected using a Zeiss LSM 710 confocal microscope (Zeiss, Oberkochen, Germany) using the Zeiss AXIO Observer Z1 microscope stand.

Chaji S., Al-Saleh J, & Gomillion C.T. (2020). Bioprinted Three-Dimensional Cell-Laden Hydrogels to Evaluate Adipocyte-Breast Cancer Cell Interactions. Gels, 6(1), 10.

+ Open protocol

+ Expand

Cell Proliferation Assay with CyQuant

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were seeded at 20,000 cells per well for the screening study or 2,000 cells per well for proliferation assays in growth medium in 96-well plates (Corning, 3603). For proliferation assays, the culture medium was removed the next day, followed by a gentle 1× wash with PBS. Treatment medium was then applied, and the cells incubated at 37 °C and 5% CO₂ until they reached ~70% confluence. Medium was then carefully aspirated, and the plate with cells attached was moved to −80 °C for at least 24 h to ensure complete cell lysis. To prepare the lysis buffer and DNA dye, CyQUANT Cell Lysis Buffer and CyQUANT GR dye (Invitrogen, C7026) were diluted in water at 1:20 and 1:400, respectively. The frozen cells were then thawed and 100 µl of the lysis buffer was added to each well. Thereafter the plate was covered to prevent light from inactivating the GR dye and was placed on an orbital shaker for 5 min before measurement. Fluorescence from each well, indicating GR dye binding to DNA, was then measured utilizing a SpectraMax M3 Microplate Reader with SoftMax Pro 5.4.2 software at an excitation wavelength of 480 nm and an emission wavelength of 520 nm.

Nwosu Z.C., Ward M.H., Sajjakulnukit P., Poudel P., Ragulan C., Kasperek S., Radyk M., Sutton D., Menjivar R.E., Andren A., Apiz-Saab J.J., Tolstyka Z., Brown K., Lee H.J., Dzierozynski L.N., He X., PS H., Ugras J., Nyamundanda G., Zhang L., Halbrook C.J., Carpenter E.S., Shi J., Shriver L.P., Patti G.J., Muir A., Pasca di Magliano M., Sadanandam A, & Lyssiotis C.A. (2023). Uridine-derived ribose fuels glucose-restricted pancreatic cancer. Nature, 618(7963), 151-158.

+ Open protocol

+ Expand

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?

Revolutionizing how scientists
search and build protocols!