GFP Expressing C57BL/6 Mouse Primary Bladder Smooth Muscle Cells

Description

GFP-Expressing Mouse Primary Bladder Smooth Muscle Cells are isolated from the bladder tissue of pathogen-free laboratory C57BL/6 mice and grown in gelatin pre-coated tissue culture flasks with Complete Growth Medium. The cells are harvested at passage 2–3 and cryopreserved in vials, supplied at 0.5 × 10⁶ cells per ml in each vial delivered frozen.

These primary cells express green fluorescent protein (GFP), which produces bright green fluorescence when exposed to blue light. GFP-expressing cells are generated through transduction with GFP-lentiviral particles. Cell identity is characterized by immunofluorescence staining with an α-smooth muscle actin antibody (A2547, Sigma).

The cells are tested negative for bacteria, yeast, fungi, and mycoplasma. Under the specified cell culture conditions, they can be expanded for 3–5 passages at a recommended split ratio of 1:2. Repeated freezing and thawing of cells are not recommended.

Specifications

Features

• Isolated from the bladder tissue of pathogen-free C57BL/6 laboratory mice
• Transduced with GFP-lentiviral particles, producing bright green fluorescence under blue light exposure
• Characterized by α-smooth muscle actin antibody immunofluorescence staining
• Harvested and cryopreserved at passage 2–3
• Supplied at 0.5 × 10⁶ cells per ml in each frozen vial
• Expandable for 3–5 passages at a split ratio of 1:2 under specified culture conditions
• Tested negative for bacteria, yeast, fungi, and mycoplasma
• Shipped frozen on dry ice overnight for immediate transfer to liquid nitrogen (−180°C)

GFP Expression

Green fluorescent protein (GFP) produces bright green fluorescence when exposed to blue light. GFP-expressing cells are developed through transducing cells with GFP-lentiviral particles.

Applications

Cell Culture Workflow

Culture Preparation

• Perform all cell culture procedures in a biosafety cabinet.
• Use aseptic techniques to prevent microbial contamination.
• Sterile-filter culture media, supplements, and reagents through a 0.2 μm filter.
• Prewarm only the amount of culture medium required right away (37°C). Use 5 ml for a T25 flask or 8–10 ml for a T75 flask when recovering, changing media, or splitting cells.
• Avoid repeated warming of complete culture medium.

Gelatin-Coated Culture Surface

• Coat sterile culture dishes or flasks with Gelatin-Based Coating Solution (Catalog No. CB6950 / 6950) for 2 minutes.
• Aspirate excess solution before seeding cells.

Recovery from a Cryovial

1. Thaw the cryovial rapidly in a 37°C water bath for less than 1 minute, or until only a small amount of ice remains.
2. Wipe the outside of the vial with 70% ethanol.
3. Transfer the cells into a sterile conical tube containing prewarmed complete medium.
4. Centrifuge the cell suspension at 200 × g for 5 minutes.
5. Carefully remove the supernatant using a sterile Pasteur pipette.
6. Resuspend the cell pellet in complete growth medium.
7. Seed the cells into a gelatin-coated flask and incubate in a humidified 37°C, 5% CO₂ incubator.
8. Leave the culture undisturbed for the initial 12–16 hours.

Primary Cell Expansion

• Wash cells with calcium- and magnesium-free 1X PBS before detachment.
• Incubate cells with prewarmed (37°C) 0.25% Trypsin-EDTA solution (Catalog No. 6914) for 3–5 minutes (2 ml for a T25 flask, 3 ml for a T75 flask).
• As soon as cells detach, add complete culture medium (FBS neutralizes trypsin) and gently pipette 1–2 times.
• Seed detached cells into fresh gelatin-coated culture vessels using the recommended split ratio of 1:2.

Follow the specified primary cell culture protocol when recovering, maintaining, expanding, or cryopreserving the cells.

Recommended Medium

Quality Control

Storage and Handling

• Cryopreserved cells are shipped frozen on dry ice by overnight delivery.
• Upon arrival, immediately transfer frozen cells to liquid nitrogen (−180°C) until ready for use.
• Primary cells can never be kept in a −20°C or −80°C freezer for long-term storage.
• Live cell shipment is available on request.
• Avoid repeated freezing and thawing of cells.
• Handle all animal cells with caution as potential pathogens, since no test procedure can completely guarantee the absence of infectious agents.

Documents

Frequently Asked Questions

Disclaimer

Investigators should handle the cells with caution and treat all animal cells as potential pathogens, since no test procedure can completely guarantee the absence of infectious agents.

For Research Use Only. Our products are not authorized for human use, for in vitro diagnostic or therapeutic procedures.