GFP Expressing C57BL/6 Mouse Primary Artery Smooth Muscle Cells

Description

Research using fluorescently labeled vascular cells requires clear information about tissue origin, strain background, reporter introduction, passage, characterization, and culture requirements. These factors are particularly important when selecting primary cells, which have a limited expansion range and require defined recovery and handling conditions.

GFP-Expressing C57BL/6 Mouse Primary Artery Smooth Muscle Cells are isolated from artery tissues of pathogen-free laboratory C57BL/6 mice. The cells provide a GFP-expressing primary smooth muscle cell model for cell culture research requiring mouse artery-derived cells and a fluorescent reporter.

GFP expression is introduced by transduction with GFP lentiviral particles. The cells are cultured in gelatin-precoated tissue culture flasks with complete smooth muscle cell growth medium and cryopreserved at passages 2–3. Each vial contains cells at a concentration of 0.5 × 10⁶ cells per mL and is delivered frozen.

Cell identity is characterized by immunofluorescence staining with an α-smooth muscle actin antibody. The cells are reported negative for bacteria, yeast, fungi, and mycoplasma. Under the specified primary cell culture conditions, they can be expanded for 3–5 passages using a recommended split ratio of 1:2.

C57-6081GFP cells are supplied for research use only. They are not authorized for human use, in vitro diagnostic procedures, or therapeutic applications.

Specifications

Features

• Primary artery smooth muscle cells isolated from laboratory C57BL/6 mice
• GFP expression introduced through GFP lentiviral particle transduction
• Cryopreserved at passages 2–3 for frozen delivery
• Supplied at a documented concentration of 0.5 × 10⁶ cells per 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 primary cell culture conditions
• Recommended 1:2 split ratio for routine expansion
• Supported by defined thawing, gelatin coating, culture, and cryopreservation protocols

Applications

These GFP-expressing C57BL/6 mouse primary artery smooth muscle cells provide a fluorescently labeled primary cell model for research workflows requiring mouse artery-derived smooth muscle cells. Application suitability should be determined according to the experimental design, the characteristics of primary cells, and the supplied culture protocol.

• Fluorescence-based observation: Visualization of GFP-expressing mouse primary artery smooth muscle cells during in vitro culture workflows.
• Primary vascular smooth muscle cell culture: Research requiring cultured C57BL/6 mouse artery-derived smooth muscle cells.
• Cell-based assays: Use of cultured primary smooth muscle cells in investigator-designed cell-based experiments.
• Immunofluorescence workflows: Fluorescence-based staining and observation of cultured primary cells using appropriately validated reagents and controls.
• FACS analysis: Analysis of cultured cells using investigator-defined fluorescence compensation, gating, and assay conditions.
• Experimental cell preparation: Expansion and preparation of primary cells for downstream experiments conducted under laboratory-validated conditions.

No disease-specific, diagnostic, therapeutic, clinical, in vivo tracking, or assay-performance claim is made. The supplied documents do not list RT-PCR, Western blotting, or immunoprecipitation as validated applications for C57-6081GFP.

Quality Control

Cell Culture Workflow

Culture Preparation

• Perform primary cell culture procedures in a biosafety cabinet.
• Use aseptic techniques to minimize 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 approximately 5 mL of medium for a T25 flask or 8–10 mL for a T75 flask when recovering 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 cryovial 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 promptly and disinfect its exterior with 70% ethanol.
3. Transfer the cell suspension into a sterile 15 mL conical tube containing 5 mL of prewarmed cell culture medium.
4. Rinse the vial 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-containing freezing medium.
6. Carefully aspirate the supernatant and resuspend the cell pellet in 5 mL of complete growth medium.
7. Seed the resuspended cells into a gelatin-coated T25 flask.
8. Place the flask in a humidified incubator at 37°C with 5% CO₂.
9. Leave the culture undisturbed for 12–16 hours.
10. Change the culture medium the following day and every 24 hours thereafter according to the current protocol.
11. Check cell confluence and morphology daily under a microscope.

Primary Cell Expansion

1. Remove the culture medium and wash the cells twice with calcium- and magnesium-free 1X PBS.
2. Add CB6914 0.25% Trypsin-EDTA Solution prewarmed to 37°C.
3. Use approximately 0.5–1 mL for a T25 flask or 2 mL for a T75 flask.
4. Incubate for 3–5 minutes while monitoring cell detachment.
5. Once the cells detach, add 5–10 mL of complete culture medium containing serum to neutralize the trypsin.
6. Gently pipette the cell suspension several times.
7. Seed the cells into fresh gelatin-coated culture vessels using a recommended split ratio of 1:2.
8. Return the culture vessels to a humidified incubator at 37°C with 5% CO₂.
9. Change the medium the following day and every 24 hours thereafter.
10. Monitor cell morphology and confluence daily.

Cryopreservation

1. Harvest the cells using the specified PBS washing and trypsinization procedure.
2. Transfer the cell suspension to a centrifuge tube and centrifuge at 200 × g for 5 minutes.
3. Carefully remove the supernatant.
4. Add 1 mL of CB6916 Freezing Medium to each cryovial and promptly resuspend the cell pellet.
5. Place the cryovials in a controlled-rate freezing container containing 100% isopropanol.
6. Freeze at −80°C for 6–12 hours as an intermediate controlled-freezing step.
7. Transfer the cryovials to a liquid nitrogen tank for long-term storage.

Storage and Handling

• Cryopreserved cells are shipped frozen on dry ice.
• Immediately transfer the cells from the dry ice shipping container to liquid nitrogen upon arrival.
• Do not use a −20°C or −80°C freezer for long-term storage of delivered cryopreserved primary cells.
• The −80°C step in the cryopreservation procedure is only an intermediate controlled-freezing step before transfer to liquid nitrogen.
• Repeated freezing and thawing of the cells are not recommended.
• Handle animal-derived cells with caution and treat them as potential biohazards.
• Follow applicable institutional biosafety and waste-disposal procedures.
• Review the supplied Safety Data Sheet before handling the product.

Authorized Use and Warranty

GFP-Expressing C57BL/6 Mouse Primary Artery Smooth Muscle Cells are distributed for research purposes only. The product is not authorized for human use, in vitro diagnostic procedures, or therapeutic procedures.

The supplier’s documented guarantee applies when the cells are used with the specified Cell Biologics medium and coating solution, cultured and tested according to the applicable online protocol, and evaluated within 35 days of product delivery.

Documents

Product Datasheet – C57-6081GFP

Primary Cell Thawing & Culture Protocol

Archived Primary Cell Culture Protocol

Cell Culture Products Safety Data Sheet 

Frequently Asked Questions