Membrite fix cell surface staining kit

Manufactured by Biotium

The MemBrite Fix Cell Surface Staining Kit is a labeling solution designed for the detection and visualization of cell surface proteins. It provides a simple and effective method for staining live cells without disrupting their native structure or function.

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Lab products found in correlation

Duolink in situ mounting medium, by Merck Group (1 mentions) Fluoromount, by Merck Group (1 mentions) Opti mem, by Thermo Fisher Scientific (1 mentions) Sp5 confocal microscope, by Leica (1 mentions) Lsm 900, by Zeiss (1 mentions) Nucblue live cell stain readyprobes reagent, by Thermo Fisher Scientific (1 mentions) Lsm900 confocal microscopy system, by Zeiss (1 mentions) Alexa fluor 594 donkey anti rabbit igg, by Cell Signaling Technology (1 mentions) Dapi staining solution, by Beyotime (1 mentions)

4 protocols using membrite fix cell surface staining kit

Chemokine receptor trafficking in HEK293 cells

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HEK293^WT; HEK293 β-arrestin 1^KO; β-arrestin 2^KO; β-arrestin 1, 2^KO cells transfected with receptors (and GFP in a T2A system or with an mGPR182-venus construct), were grown on MatTek slides. These were incubated with 100 nM chemokines (all labeled in AF647, ATTO565 or ATTO700) for 45 minutes at 37°C. Cells were washed with PBS, fixed in 4% paraformaldehyde (PFA), permeabilized, stained with antibodies, and mounted in either Fluoromount (Sigma, Cat: F4680) or DAPI-containing Duolink in situ mounting medium (Sigma, Cat: DUO82040). Membranes were visualized either by staining with MemBrite Fix Cell Surface Staining Kit (Biotium) following manufacturer’s instructions, or by transiently transfecting cells with a plasmid expressing the myristoylation/palmitylation motif from the Lck fused to the N-terminus of mCherry generating a plasma membrane marker (13 (link)).
For live imaging experiments, HEK293 expressing mGPR182 GFP were grown on MatTek slides. On the day of the experiment, complete medium was replaced with Optimem (Gibco) containing 10% FBS. CCL20-AF647 was added dropwise during live acquisition at t = 1 minute, at a final concentration of 200 nM. Live imaging was performed using a Leica SP5 confocal microscope. Chemokine uptake was quantified using ImageJ by measuring Integrated Density (set to threshold) on selected ROIs.

Melgrati S., Gerken O.J., Artinger M., Radice E., Szpakowska M., Chevigné A., D’Uonnolo G., Antonello P., Thelen S., Pelczar P., Legler D.F, & Thelen M. (2023). GPR182 is a broadly scavenging atypical chemokine receptor influencing T-independent immunity. Frontiers in Immunology, 14, 1242531.

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Visualizing Cleaved Gasdermin D in RAW 264.7 Cells

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Before washing the RAW 264.7 cells, the cell nucleus was stained by using NucBlue™ Live cell stain ReadyProbes reagent (Hoechst33342, Invitrogen Cat. No. 2325910). Harvested cells were washed with cold PBS twice and the cell surface membrane was stained with MemBrite fix Cell Surface staining kit (Biotium, cat. No. 30093) according to the company’s instruction. Cells were rinsed with PBS and fixed with 4% methanol for 15 min at room temperature. After being washed with PBS 3 times, cells were blocked with PBS/5% BSA for 1 h at room temperature. Subsequently, cells were incubated with primary antibody against cleaved GSDMD (dilution 1:200; Cat. No. 101375, Cell Signaling Technology) overnight at 4 °C. Goat anti-rabbit IgG (dilution 1:250; Cat. No. D00304-15, LI-COR) served as a secondary antibody. Hoechst3334 was used for DNA dye. Cells were visualized by using Axio Observer.Z1/7 equipped with Zeiss LSM900 confocal microscopy system. The z-stack images of cells were acquired with Plan-Apochromat 63x/1.40 Oil DIC M27 objective lens. SR-4Y fast acquisition mode of Airyscan and 4× averaging was used. The images obtained by confocal microscope were merged and combined by FIJI Image J (28 (link)).

Tan C., Reilly B., Jha A., Murao A., Lee Y., Brenner M., Aziz M, & Wang P. (2022). Active Release of eCIRP via Gasdermin D Channels to Induce Inflammation in Sepsis. Journal of immunology (Baltimore, Md. : 1950), 208(9), 2184-2195.

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Visualizing p-MLKL in LPS-induced Cell Death

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RAW cells were cultured on glass bottom petri dishes and treated with LPS and z-VAD-fmk. The nuclei were stained by using NucBlue^TM Live cell stain ReadyProbes reagent (Hoechst 33342, Cat. No. 2325910, Invitrogen,) and then rinsed twice with cold PBS. The cell surface membrane was then stained with MemBrite fix Cell Surface staining kit (Biotium, Cat. No. 30093) according to the company’s instructions. Cells were washed with PBS and fixed using 4% methanol for 15 minutes at room temperature. The solution was removed, and the cells were washed three times with PBS and blocked with 5% BSA in PBS for 1 hour at room temperature. After blocking, cells were incubated in rabbit anti-p-MLKL primary antibody (dilution 1:200; Cat. No. 101375, Cell Signaling Technology) overnight at 4^oC. Cells were washed three times and incubated in Alexa Fluor 594 Donkey anti-rabbit IgG (Cat. No. A21207, Thermo Fisher Scientific) for 2 hours. After washing three times, the samples were sealed using prolong gold anti-fade (Cat. No. P36934, Thermo Fisher Scientific). Cells were visualized by using Axio Observer. Z1/7 equipped with Zeiss LSM900 confocal microscopy system. Z-stack images were acquired with Plan-Apochromat 63x/1.40 Oil DIC M27 objective lens. SR-4Y fast acquisition mode of Airyscan and 4× averaging was used. The images obtained by confocal microscope were merged and combined by FIJI Image J.

Reilly B., Tan C., Murao A., Nofi C., Jha A., Aziz M, & Wang P. (2022). Necroptosis-Mediated eCIRP Release in Sepsis. Journal of Inflammation Research, 15, 4047-4059.

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Quantification of FMDV Cell Binding and Internalization

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For cell binding assays, IBRS-2 or PK-15 cells seeded on glass bottom cell culture dishes were incubated for 1 h at 4°C with equivalent amounts of FMDV particles. After extensive washes with cold PBS, the cells were fixed for 3 min with methanol at -20°C and analyzed by immunofluorescence with a rabbit antibody against the VP1 viral protein followed by Alexalabeled secondary antibodies. The surface of cells stained with MemBrite Fix Cell Surface Staining Kit (Biotium, 30093-T) and nuclei were labeled with DAPI Staining Solution (Beyotime, C1006). The cells were collected and quantified by qPCR. To quantify virus uptake, IBRS-2 or PK-15 cells seeded on glass bottom cell culture dishes were incubated with equal amounts of FMDV virus particles for 30 min at 37°C. Then, the cells were incubated with trypsin-EDTA for 5 min at 37°C to remove noninternalized particles and fixed with 4% PFA for 15 min at room temperature. After permeabilization with 0.1% Triton-100 for 5 min, the internalized particles were immunolabeled with rabbit anti-VP1 antibody followed by an Alexa Fluor 488-conjugated anti-rabbit antibody. The cells were collected and quantified by qPCR. Another group of cells were infected under the same conditions and RNA was extracted for subsequent qPCR detection.

Peng G., Liu T., Qi X., Wang Y., Ren J., Peng J., Du X., Hu S., Wu S., Zhao Y., Li D, & Zheng H. (2024). A genome-wide CRISPR screening uncovers that TOB1 acts as a key host factor for FMDV infection via both IFN and EGFR mediated pathways. PLOS Pathogens, 20(3), e1012104.

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