RFP Expressing C57BL/6 Mouse Primary Stomach Smooth Muscle Cells

Description

Identifying a defined cell population during fluorescence imaging or mixed-culture observation can require additional labeling steps that add time and variability to primary cell experiments. Researchers working with mouse gastric smooth muscle cells may therefore need a tissue-specific primary cell model that already expresses a fluorescent marker and is supplied with documented culture guidance.

RFP-Expressing C57BL/6 Mouse Primary Stomach Smooth Muscle Cells are isolated from the stomach tissues of pathogen-free laboratory C57BL/6 mice. These mouse primary gastric smooth muscle cells are grown in gelatin pre-coated tissue culture flasks with Complete Growth Medium and supplied as cryopreserved cells.

RFP-expressing cells are developed by transduction with RFP lentiviral particles. Red fluorescent protein produces red fluorescence when exposed to suitable yellow-green excitation, supporting direct visualization of the labeled cells under compatible fluorescence imaging conditions.

Cells at passages 2–3 are harvested and cryopreserved in vials. Each vial contains 0.5 × 10⁶ cells per mL and is delivered frozen. Cell identity is characterized by immunofluorescent staining with an α-smooth muscle actin antibody (A2547, Sigma).

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

For Research Use Only. Not for human use, in vitro diagnostic procedures, or therapeutic applications.

Specifications

Features

• Primary gastric smooth muscle cells isolated from stomach tissues of pathogen-free C57BL/6 mice
• RFP expression introduced through transduction with RFP lentiviral particles
• Supplied cryopreserved at passages 2–3
• Each vial contains 0.5 × 10⁶ cells per mL
• Characterized by α-smooth muscle actin immunofluorescent staining
• Reported negative for bacteria, yeast, fungi, and mycoplasma
• Recommended for adherent culture on gelatin-coated culture vessels
• Reported expansion of 2–3 passages at a split ratio of 1:2 under specified conditions
• Shipped frozen on dry ice for immediate transfer to liquid nitrogen

Applications

These RFP-expressing mouse primary stomach smooth muscle cells provide a fluorescently labeled primary cell model for research requiring visualization of gastric smooth muscle cells under compatible in vitro culture conditions.

• Fluorescence imaging of RFP-expressing primary smooth muscle cells
• Visualization and tracking of labeled mouse gastric smooth muscle cells in culture
• In vitro mouse stomach smooth muscle cell research
• Observation of primary smooth muscle cell morphology and culture behavior
• Identification of RFP-labeled cells in compatible mixed-cell or co-culture experiments
• Immunofluorescence-based observation of cultured primary smooth muscle cells

Suitability for a specific assay or experimental system should be determined by the investigator. No application-specific performance data are provided in the available product documents.

Quality Control

Primary Cell Culture Guidance

Before Starting

• Perform all cell culture procedures in a biosafety cabinet.
• Use aseptic technique to minimize microbial contamination.
• Sterile filter culture media, supplements, and reagents through a 0.2 µm filter.
• Prewarm only the volume of culture medium required for immediate use to 37°C.
• Avoid repeatedly warming complete culture medium.

Gelatin Coating

• Coat sterile culture dishes or flasks with CB6950 Gelatin-Based Coating Solution for 2 minutes.
• Aspirate the excess coating 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 few ice pieces remain.
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 culture medium.
4. Rinse the vial with an additional 0.5–1 mL of medium to recover 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 prewarmed growth medium.
7. Seed the resuspended cells into a T25 flask pre-coated with CB6950 Gelatin-Based Coating Solution.
8. Place the flask in a humidified incubator at 37°C with 5% CO₂ and leave the culture undisturbed for 12–16 hours.
9. Change the medium on the second day and every 24 hours thereafter according to the current culture protocol.
10. Check cell confluence and morphology daily under a microscope.

Primary Cell Expansion

• Remove the culture medium and wash the adherent cells twice with room-temperature 1X PBS without calcium or magnesium.
• Incubate the cells with prewarmed 0.25% Trypsin-EDTA Solution (Catalog No. CB6914) at 37°C for 3–5 minutes.
• The current protocol specifies 0.5–1 mL of Trypsin-EDTA for a T25 flask and 2 mL for a T75 flask.
• Once the cells detach, add culture medium containing serum to neutralize the trypsin.
• Seed the cells into fresh gelatin-coated flasks or plates.
• Use a recommended split ratio of 1:2.
• Return the culture vessels to a humidified incubator at 37°C with 5% CO₂.

Preparation for Experiments

• Monitor the cells daily for confluence and appropriate morphology.
• The protocol notes that confluent cells 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 pre-wash the cells twice with 1X PBS between medium changes during this period.
• Before an experiment, the protocol recommends filtering the cells through a 40 µm cell strainer to remove cell clumps and most large dead or floating cells.

This section is a condensed summary. Follow the current Primary Cell Culture Protocol in the Documents section for complete handling instructions.

Storage and Handling

• Cryopreserved cells are shipped frozen on dry ice overnight.
• Upon receipt, transfer the frozen vial from the dry ice shipping container to liquid nitrogen immediately.
• Do not use a −20°C or −80°C freezer for routine long-term storage of the supplied primary cells.
• Avoid repeated freeze-thaw cycles.
• Treat animal-derived cells as potentially biohazardous and follow appropriate laboratory safety procedures.
• Use only for laboratory research. The cells are not authorized for human, clinical, diagnostic, veterinary, or therapeutic use.

Documents

Cell-Culture-Protocol-CB

1708462493

1698246847

The first two documents contain primary cell culture guidance. The third document contains safety information for cell culture media, reagents, and primary cells.

Frequently Asked Questions