GFP Expressing C57BL/6 Mouse Primary Small Intestinal Smooth Muscle Cells

Description

GFP-Expressing Mouse Primary Small Intestinal Smooth Muscle Cells from Cell Biologics are isolated from the small intestines of pathogen-free laboratory C57BL/6 mice and grown in gelatin pre-coated tissue culture flasks with Complete Growth Medium.

GFP-Expressing C57BL/6 Mouse Primary Small Intestinal Smooth Muscle Cells provide a cryopreserved primary smooth muscle cell model isolated from the small intestines of pathogen-free laboratory C57BL/6 mice. The cells express green fluorescent protein generated through GFP-lentiviral particle transduction and produce bright green fluorescence when exposed to blue light.

These mouse primary small intestinal smooth muscle cells are characterized by immunofluorescence staining with an α-smooth muscle actin antibody. Cells are harvested at passage 2–3, supplied frozen at 0.5 × 10⁶ cells per milliliter in each vial, and can be expanded for 3–5 passages at a recommended split ratio of 1:2 under the specified primary cell culture conditions.

A defined workflow covering cryovial recovery, gelatin-coated culture, primary cell expansion, routine medium changes, subculture, and cryopreservation is available to support consistent handling of these GFP-expressing mouse intestinal smooth muscle cells.

Specifications

Features

• GFP-expressing mouse primary small intestinal smooth muscle cells

• Isolated from the small intestines of pathogen-free laboratory C57BL/6 mice

• Stable GFP-expressing cells generated through GFP-lentiviral particle transduction

• Produces bright green fluorescence when exposed to blue light

• Characterized by α-smooth muscle actin immunofluorescence staining

• Harvested and cryopreserved at passage 2–3

• Supplied at 0.5 × 10⁶ cells/ml per vial

• Expandable for 3–5 passages under the specified culture conditions

• Recommended split ratio of 1:2

• Tested negative for bacteria, yeast, fungi, and mycoplasma

• Supported by a primary cell recovery, expansion, and freezing protocol

GFP Expression and Cell Identification

The green fluorescent protein expressed by these cells produces bright green fluorescence when exposed to blue light. This built-in fluorescent signal allows the GFP-expressing mouse primary smooth muscle cells to be visually identified under compatible fluorescence-based observation.

• Stable GFP-expressing cells generated by lentiviral particle transduction

• Bright green fluorescent signal under blue-light excitation

• Supports visual identification during primary cell culture

• May assist with identifying GFP-positive cells during staining or cell-based experimental procedures

Applications

• Mouse Intestinal Smooth Muscle Cell Research

  Provides a primary smooth muscle cell model derived specifically from the small intestines of C57BL/6 mice for in vitro research involving mouse intestinal smooth muscle cells.

• Primary Smooth Muscle Cell Culture

  Suitable for researchers requiring cryopreserved primary mouse smooth muscle cells that can be recovered, maintained, and expanded using gelatin-coated culture vessels and complete smooth muscle cell medium.

• GFP Fluorescence Observation

  Bright green fluorescence under blue-light exposure supports visualization of GFP-expressing intestinal smooth muscle cells in compatible fluorescence microscopy workflows.

• α-Smooth Muscle Actin Characterization

  The cells are characterized by immunofluorescence staining with an α-smooth muscle actin antibody, providing a documented smooth muscle cell identification reference.

• Immunofluorescence and Cell Staining Workflows

  Suitable for laboratory workflows requiring cultured primary cells for staining, microscopy, and other designed cell-based experiments performed under the recommended culture conditions.

• FACS Sample Preparation

  The supplied primary cell culture protocol includes guidance for preparing cultured cells before FACS analysis, including removal of cell clumps with a 40 μm cell strainer.

• Primary Cell Expansion Studies

  Cells can be expanded for 3–5 passages using a recommended 1:2 split ratio, supporting experiments that require cultured cells across a limited primary cell expansion window.

• Cryopreserved Primary Cell Recovery

  Suitable for laboratories requiring a defined recovery workflow covering rapid thawing, residual DMSO removal, gelatin-coated seeding, daily observation, and subsequent expansion.

Specific disease, therapeutic, contraction, migration, molecular, or drug-screening applications have not been established in the supplied product documentation. Researchers should validate suitability for their intended experimental design.

Cell Culture Workflow

Culture Preparation

• Perform all primary 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 volume of complete cell culture medium required for immediate use.

• Avoid repeated warming of complete culture medium.

• Use approximately 5 ml of medium for a T25 flask and 8–10 ml for a T75 flask when recovering, changing medium, or splitting cells.

Gelatin-Coated Culture Surface

• Use sterile tissue culture dishes or flasks coated with a gelatin-based coating solution (Cell Biologics, Catalog No. CB6950).

• Apply the gelatin-based coating solution for approximately 2 minutes.

• Aspirate excess coating solution before seeding the cells.

• Seed recovered or passaged cells only after the culture vessel has been prepared.

Cell Recovery from Cryovial

1. Quickly thaw the cryovial in a 37°C water bath for less than 1 minute or until only a few ice pieces remain.

2. Immediately remove the vial and wipe the exterior with 70% ethanol.

3. Transfer the cells into a sterile 15 ml conical tube containing 5 ml of prewarmed cell culture medium.

4. Rinse the cryovial with an additional 0.5–1 ml of medium to recover remaining cells.

5. Centrifuge the cells at 200 × g for 5 minutes to remove residual DMSO.

6. Carefully aspirate the supernatant and resuspend the cell pellet in 5 ml of complete cell culture growth medium.

7. Transfer the resuspended cells into a gelatin-coated T25 flask.

8. Culture the cells in a humidified incubator at 37°C with 5% CO₂.

9. Leave the newly seeded culture undisturbed for approximately 12–16 hours.

10. Change the medium on the second day and every 24–48 hours thereafter.

11. Check the cells daily for confluence and appropriate cell morphology.

Primary Cell Expansion

1. Remove the culture medium from the flask.

2. Wash the cells twice with room-temperature 1X PBS without calcium and magnesium.

3. Add prewarmed 0.25% Trypsin-EDTA solution (0.5–2 ml for a T25 flask, 2.0–3.0 ml for a T75 flask) and incubate at 37°C for approximately 3–5 minutes.

4. Once the cells have detached, immediately add complete culture medium containing fetal bovine serum to neutralize the trypsin.

5. Gently pipette the cell suspension several times.

6. Seed the cells into fresh gelatin-coated culture vessels.

7. Return the culture vessels to a humidified 37°C, 5% CO₂ incubator.

8. Use the recommended split ratio of 1:2.

9. Continue daily microscopic checks for confluence and appropriate morphology.

Routine Culture Guidance

• Change culture medium the following day and then every 24–48 hours afterwards (change medium daily when cells are >70–80% confluent).

• Use approximately 5 ml of medium for a T25 flask and 10 ml for a T75 flask.

• Before each medium change, pre-wash the cells twice with 1X PBS without calcium and magnesium.

• Monitor the cells daily under a microscope.

• Avoid repeated freezing and thawing of the cells.

Preparation for Cell-Based Experiments

• Once the cells reach confluence, the protocol permits an additional 24-hour culture period before cell-based assays, staining, FACS analysis, or other designed experiments.

• Continue daily medium changes during this period.

• Pre-wash the cells twice with 1X PBS between medium changes.

• Before an experiment, a 40 μm cell strainer may be used to remove cell clumps and large dead or floating cells.

Cryopreservation

• Wash the adherent cell layer with calcium- and magnesium-free 1X PBS before detachment.

• Detach cells using prewarmed 0.25% Trypsin-EDTA for approximately 3–5 minutes.

• Add complete culture medium to neutralize the trypsin and gently pipette 1–2 times.

• Centrifuge the collected cell suspension at 200 × g for 5 minutes.

• Carefully remove the supernatant and quickly resuspend the cell pellet in 1 ml of freezing buffer (Cell Biologics, Catalog No. CB6916) per cryovial.

• Place vials in a Nalgene "Mr. Frosty" freezing container filled with 100% isopropanol and freeze at -80°C for 6–12 hours.

• Transfer the frozen cryovials to a liquid nitrogen tank (-180°C) for long-term storage.

Storage and Handling

• Cryopreserved cells are shipped frozen on dry ice.

• Immediately transfer the frozen vial to liquid nitrogen storage upon arrival.

• Store in 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.

• Avoid repeated freeze-thaw cycles.

• Handle all animal-derived cells with appropriate laboratory precautions.

• Treat animal cells as potential biological hazards because no testing procedure can guarantee the complete absence of infectious agents.

• For Research Use Only. Not intended for human use, diagnostic procedures, or therapeutic applications.

Quality Control

Documents

C57-6088GFP Product Information

Primary Cell Culture Protocol

Additional Cell Culture Protocol

Safety Data Sheet

Frequently Asked Questions