GFP Expressing C57BL/6 Mouse Primary Esophageal Smooth Muscle Cells

Description

Fluorescence-based cell experiments require a traceable cell population while retaining the defined tissue, cell type, and strain identity of the primary model. GFP-Expressing Mouse Primary Esophageal Smooth Muscle Cells provide a fluorescently identifiable C57BL/6 mouse esophageal smooth muscle cell model for primary cell culture and cell-based experimental workflows.

These mouse primary esophageal smooth muscle cells are isolated from the esophageal tissues of pathogen-free laboratory C57BL/6 mice and cultured in gelatin-precoated tissue culture flasks with Cell Biologics' Complete Growth Medium. GFP-expressing cells are developed through transduction with GFP-lentiviral particles. Green fluorescent protein produces bright green fluorescence when exposed to blue light.

Cells are harvested and cryopreserved at passages 2–3. The product datasheet states that each vial contains cells at a concentration of 0.5 × 10⁶ cells/mL and is delivered frozen. Cell identity is characterized by immunofluorescence staining with an α-smooth muscle actin antibody (A2547, Sigma).

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

Specifications

Important: The datasheet documents a concentration of 0.5 × 10⁶ cells/mL but does not state the total vial volume. Confirm the total cells per vial separately when required for experimental planning.

Features

• Primary esophageal smooth muscle cells isolated from C57BL/6 mouse esophageal tissue
• GFP expression introduced through GFP-lentiviral particle transduction
• Bright green fluorescence under blue-light excitation
• Harvested and cryopreserved at passages 2–3
• Documented concentration of 0.5 × 10⁶ cells/mL
• Characterized by α-smooth muscle actin immunofluorescence staining
• Reported negative for bacteria, yeast, fungi, and mycoplasma
• Expandable for 3–5 passages under the specified culture conditions
• Recommended split ratio of 1:2
• Adherent growth on gelatin-precoated culture vessels

Applications

These GFP-expressing mouse primary esophageal smooth muscle cells support laboratory workflows that require a fluorescently identifiable primary smooth muscle cell population with defined C57BL/6 strain and esophageal tissue origin.

• GFP-based cell observation: Visualize GFP-expressing primary cells under appropriate blue-light excitation.
• Fluorescence-based cell assays: Use a fluorescently identifiable primary esophageal smooth muscle cell population in designed cell-based experiments.
• Primary esophageal smooth muscle cell culture: Study cultured mouse esophageal smooth muscle cells under the supplied growth and subculture conditions.
• Immunofluorescence workflows: Use cultured cells in staining workflows; product identity is characterized using α-smooth muscle actin immunofluorescence.
• FACS sample preparation: The supplied culture protocol includes sample preparation guidance for FACS analysis.
• Cell-based experimental workflows: Prepare confluent cultures for staining, FACS analysis, or other designed experiments following the supplied protocol.

Suitability for a specific assay should be established by the investigator under the intended experimental conditions.

Recommended Culture Materials

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 for immediate use to 37°C.
• Use 5 mL of culture medium for a T25 flask or 8–10 mL for a T75 flask when recovering cryopreserved cells, changing medium, or splitting cells.
• Avoid repeated warming of complete culture medium.

Gelatin Coating

1. Add CB6950 Gelatin-Based Coating Solution to a sterile culture flask or dish.
2. Allow the coating solution to contact the culture surface for 2 minutes.
3. Aspirate the excess coating solution before seeding cells.

Recovery from a Cryovial

1. Thaw the vial rapidly in a 37°C water bath for less than 1 minute or until only a small amount of ice remains.
2. Remove the vial immediately and disinfect its exterior with 70% ethanol.
3. Transfer the cells into a sterile 15 mL conical tube containing 5 mL of prewarmed Cell Biologics culture medium.
4. Rinse the vial with an additional 0.5–1 mL of medium to collect remaining cells.
5. Centrifuge at 200 × g for 5 minutes to remove residual DMSO.
6. Carefully aspirate the supernatant and resuspend the cell pellet in 5 mL of Cell Biologics Cell Culture Growth Medium.
7. Seed the resuspended cells into a gelatin-precoated T25 flask.
8. Incubate in a humidified incubator at 37°C with 5% CO₂ and leave the culture undisturbed for 12–16 hours.
9. Change the medium the following day and every 24 hours thereafter according to the current supplied protocol.
10. Check cell confluence and morphology daily under a microscope.

Primary Cell Expansion

1. Remove the culture medium and wash the cells twice with room-temperature 1X PBS without calcium and magnesium.
2. Incubate the cells with CB6914 0.25% Trypsin-EDTA, prewarmed to 37°C, for 3–5 minutes.
3. Use 0.5–1 mL of Trypsin-EDTA for a T25 flask or 2 mL for a T75 flask.
4. As soon as the cells detach, add 5–10 mL of complete culture medium containing FBS to neutralize the trypsin.
5. Gently pipette the cell suspension several times.
6. Seed the cells into fresh gelatin-precoated culture vessels.
7. Return the cultures to a humidified incubator at 37°C with 5% CO₂.
8. Use the recommended split ratio of 1:2.

Preparation for Cell-Based Experiments

• After cells reach confluence, cultures may be allowed to overgrow for up to 24 hours before cell-based assays, staining, FACS analysis, or other designed experiments.
• Change the medium daily and wash the cells twice with 1X PBS between medium changes.
• Before an experiment, the protocol recommends passing cells through a 40 μm cell strainer to remove cell clumps and most dead, floating large cells.

Cell Freezing

1. Harvest the cells using the washing, trypsinization, and centrifugation procedure described in the culture protocol.
2. Add 1 mL of CB6916 Freezing Buffer to each vial and rapidly resuspend the cell pellet.
3. Place the vials in a Nalgene "Mr. Frosty" freezing container containing 100% isopropanol.
4. Freeze at -80°C for 6–12 hours as an intermediate controlled-freezing step.
5. Transfer the vials to a liquid nitrogen tank for long-term storage.

The -80°C step described above is an intermediate freezing procedure only. Received cryopreserved cells should be transferred immediately to liquid nitrogen and must not be routinely stored at -80°C.

Quality Control

Shipping, Storage, and Handling

• Cryopreserved cells are shipped frozen on dry ice by overnight delivery.
• Upon arrival, transfer the frozen vial immediately to liquid nitrogen (-180°C) until ready for use.
• Do not use a -20°C or -80°C freezer for routine storage of received primary cells.
• Live-cell shipment is available on request according to the product datasheet.
• Avoid repeated freezing and thawing.
• Handle all animal cells with caution and treat them as potential pathogens because no testing procedure can guarantee the complete absence of infectious agents.

Warranty Requirements

The manufacturer's warranty applies only when Cell Biologics cells are purchased and used with the appropriate Cell Biologics medium and coating solution, cultured and tested according to the supplied protocols, and evaluated within 35 days of product delivery.

Documents

C57-6089GFP Product Datasheet

Primary Cell Culture Protocol

Archived Primary Cell Culture Protocol

Cell Culture Products Safety Data Sheet

Frequently Asked Questions

What are GFP-Expressing Mouse Primary Esophageal Smooth Muscle Cells?

They are primary smooth muscle cells isolated from the esophageal tissues of pathogen-free laboratory C57BL/6 mice. GFP expression is introduced through GFP-lentiviral particle transduction, and the cells are supplied as a cryopreserved vial.

How does this product differ from cells isolated from a GFP-transgenic mouse?

The C57-6089GFP datasheet states that GFP-expressing cells are developed through transduction with GFP-lentiviral particles. The product is therefore described according to that documented preparation method rather than as cells isolated from a GFP-transgenic mouse.

How is GFP introduced into these primary cells?

According to the product datasheet, GFP expression is introduced through transduction with GFP-lentiviral particles. GFP produces bright green fluorescence when exposed to blue light.

At what passage are the cells cryopreserved?

The cells are harvested and cryopreserved at passages 2–3.

How many cells are supplied?

The product datasheet states that each vial contains cells at a concentration of 0.5 × 10⁶ cells/mL. The datasheet does not specify the total vial volume, so the total cells per vial should be confirmed separately when required.

How are the mouse primary esophageal smooth muscle cells characterized?

Cell identity is characterized by immunofluorescence staining with an α-smooth muscle actin antibody, A2547 from Sigma.

What contamination testing is documented?

The product datasheet reports negative results for bacteria, yeast, fungi, and mycoplasma.

How many passages can the cells be expanded?

Under the specified Cell Biologics culture conditions, the cells can be expanded for 3–5 passages at a recommended split ratio of 1:2.

What medium and coating solution are recommended?

The suggested medium is M2268 Complete Smooth Muscle Cell Medium with Kit, 500 mL. The current culture protocol specifies CB6950 Gelatin-Based Coating Solution for coating culture vessels.

What culture conditions are recommended?

Cells are cultured in a humidified incubator at 37°C with 5% CO₂ on gelatin-precoated culture vessels. The current protocol recommends checking cell confluence and morphology daily.

Can the cells be stored at -80°C after arrival?

No. Upon arrival, transfer the frozen vial immediately to liquid nitrogen. The -80°C step in the freezing protocol is only a 6–12-hour intermediate controlled-freezing step before transfer to liquid nitrogen.

Are these cells intended for clinical, diagnostic, or therapeutic use?

No. The cells are for research use only and are not authorized for human use, in vitro diagnostic procedures, or therapeutic procedures.