GFP Expressing C57BL/6 Mouse Primary Prostate Smooth Muscle Cells

Description

Fluorescence-based primary cell experiments require a defined cell model with a documented tissue source, cell identity, passage, and culture workflow. These GFP-expressing C57BL/6 mouse primary prostate smooth muscle cells provide a cryopreserved prostate-derived smooth muscle cell preparation with documented GFP introduction and α-smooth muscle actin characterization.

GFP-Expressing Mouse Primary Prostate Smooth Muscle Cells are isolated from the prostate tissues of pathogen-free laboratory C57BL/6 mice and grown in gelatin pre-coated tissue culture flasks with Complete Growth Medium. GFP-expressing cells are developed through transducing GFP-lentiviral particles. Green fluorescent protein produces bright green fluorescence when exposed to blue light.

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 immunofluorescence staining with an α-smooth muscle actin antibody (A2547, Sigma).

The cells are documented as 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. Repeated freezing and thawing are not recommended.

Specifications

Features

• Primary prostate smooth muscle cells isolated from pathogen-free laboratory C57BL/6 mice
• GFP expression introduced through transduction with GFP-lentiviral particles
• Bright green GFP fluorescence when exposed to blue light
• Harvested and cryopreserved at passages 2–3
• Supplied at 0.5 × 10⁶ cells per mL in a cryopreserved vial
• Characterized by α-smooth muscle actin immunofluorescence staining
• Documented as negative for bacteria, yeast, fungi, and mycoplasma
• Expandable for 3–5 passages at a recommended split ratio of 1:2 under the specified conditions
• Supported by a defined thawing, gelatin coating, expansion, and cryopreservation protocol

Applications

These GFP-expressing mouse primary prostate smooth muscle cells are intended for research workflows that require prostate-derived primary smooth muscle cells with GFP fluorescence and documented α-smooth muscle actin characterization.

• GFP fluorescence-based cell observation
• Primary prostate smooth muscle cell culture studies
• Immunofluorescence staining workflows
• FACS analysis
• Cell-based assays
• Primary cell expansion under the specified culture conditions
• Preparation of cultured cells for designed research experiments

The supplied documentation does not provide application-specific performance data, GFP-positive percentages, viability specifications, or disease-model validation.

Cell Culture Workflow

Culture Preparation

• Perform all cell culture procedures in a biosafety cabinet.
• Use aseptic techniques to prevent microbial contamination.
• Sterile filter all 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 medium for a T25 flask and 8–10 mL for a T75 flask when recovering cryopreserved cells, changing medium, or splitting cells.
• Repeated prewarming of complete culture medium is not recommended.

Gelatin Coating

1. Coat sterile culture dishes or flasks with Gelatin-Based Coating Solution (Catalog No. CB6950).
2. Allow the coating solution to remain in contact with the culture surface for 2 minutes.
3. Aspirate the excess coating solution before seeding cells.

Recovery of Cryopreserved Cells

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. Immediately remove the vial 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 Cell Culture Growth Medium.
7. Seed the resuspended cells into a gelatin-coated T25 flask.
8. Incubate at 37°C with 5% CO₂ and leave the culture undisturbed for 12–16 hours.
9. Change the medium on the following day and every 24 hours thereafter.
10. Check cell confluence and morphology daily under a microscope.

Primary Cell Expansion

1. Remove the culture medium and wash the cells twice with 1X phosphate-buffered saline without calcium or magnesium.
2. Incubate cells with prewarmed 0.25% Trypsin-EDTA Solution (Catalog No. CB6914) 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. Once the cells detach, immediately add 5–10 mL of culture medium containing fetal bovine serum to neutralize the trypsin.
5. Gently pipette the suspension up and down several times.
6. Seed cells into fresh gelatin-coated flasks or plates using the recommended 1:2 split ratio.
7. Return the culture vessels to a humidified 37°C, 5% CO₂ incubator.
8. Change the medium on the following day and every 24 hours thereafter.
9. Check cell confluence and morphology daily.

Preparation for Experiments

• Once cells reach confluence, cultures may be allowed to overgrow for 24 hours before cell-based assays, staining, FACS analysis, or designed experiments.
• Change the medium daily and pre-wash cells twice with 1X PBS between medium changes.
• Before an experiment, cells may be filtered through a 40 μm cell strainer (BD 352340) to remove cell clumps and most dead or floating large cells.

Freezing Cultured Cells

1. Harvest and centrifuge cultured cells at 200 × g for 5 minutes.
2. Quickly resuspend the cell pellet using 1 mL of Freezing Medium (Catalog No. CB6916) per vial.
3. Place the vials in a Nalgene “Mr. Frosty” freezing container filled with 100% isopropanol.
4. Hold at −80°C for 6–12 hours as part of the controlled freezing procedure.
5. Transfer the frozen vials to a liquid nitrogen tank for long-term storage.

The temporary −80°C step applies only to the controlled freezing procedure for cultured cells. Received cryopreserved product should be transferred directly from dry ice to liquid nitrogen storage.

Quality Control

Storage and Handling

• Cryopreserved cells are shipped frozen on dry ice.
• Upon receipt, immediately transfer the frozen vial to a liquid nitrogen storage tank until use.
• Do not keep received primary cells in a −20°C or −80°C freezer for routine or long-term storage.
• Repeated freezing and thawing are not recommended.
• Treat all animal cells as potential biohazards because no testing procedure can completely guarantee the absence of infectious agents.
• Use appropriate laboratory safety procedures when handling the cells.

Product Support Conditions

The documented product guarantee applies when the cells are purchased and used with the specified cell culture medium and coating solution, cultured according to the supplied protocol, and tested within 35 days of delivery.

Documents

C57-6041GFP Product Datasheet

Primary Cell Culture Protocol

Supplementary Primary Cell Culture Protocol

Cell Culture Products Safety Data Sheet

Frequently Asked Questions

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

They are primary smooth muscle cells isolated from the prostate tissues of pathogen-free laboratory C57BL/6 mice. The cells are developed through transduction with GFP-lentiviral particles and supplied as a cryopreserved preparation.

Are these primary or immortalized prostate smooth muscle cells?

They are primary mouse prostate smooth muscle cells. The supplied product documentation does not describe them as immortalized cells.

How is GFP introduced into the cells?

According to the product datasheet, GFP-expressing cells are developed through transducing GFP-lentiviral particles.

What fluorescence is produced by GFP?

The product datasheet states that green fluorescent protein gives rise to bright green fluorescence when exposed to blue light.

At what passage are the cells cryopreserved?

Cells at passages 2–3 are harvested from culture flasks and cryopreserved in vials.

How many cells are supplied?

Each cryopreserved vial contains 0.5 × 10⁶ cells per mL and is delivered frozen.

How are the mouse prostate smooth muscle cells characterized?

The cells are characterized by immunofluorescence staining with an α-smooth muscle actin antibody (A2547, Sigma).

What microbial testing is documented?

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

How many passages can the primary cells be expanded?

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

What medium is suggested for culturing these cells?

The suggested medium is M2268 Complete Smooth Muscle Cell Medium w/ Kit, supplied in a 500 mL format.

Is a coated culture vessel required?

The supplied protocol directs users to coat sterile culture vessels with CB6950 Gelatin-Based Coating Solution for 2 minutes and aspirate the excess solution before seeding cells.

How should the cryopreserved cells be thawed?

Thaw the vial rapidly in a 37°C water bath for less than 1 minute or until only a small amount of ice remains. Disinfect the vial, transfer the cells into prewarmed medium, and centrifuge at 200 × g for 5 minutes to remove residual DMSO before seeding into a gelatin-coated T25 flask.

How often should the culture medium be changed?

The current supplied protocol directs users to change the medium on the day following cell recovery and every 24 hours thereafter. Cell confluence and morphology should be checked daily.

Can the received vial be stored at −80°C?

No. Upon receipt, transfer the vial immediately from the dry ice shipping container to liquid nitrogen storage. The −80°C step described in the freezing protocol is only a temporary controlled freezing step for cultured cells before transfer to liquid nitrogen.

Can these cells be repeatedly frozen and thawed?

No. Repeated freezing and thawing of the primary cells are not recommended.

What research workflows are described in the supplied protocol?

The protocol refers to cell-based assays, staining, FACS analysis, and other designed experiments following appropriate culture preparation. It does not provide application-specific performance or validation data.

Are these cells intended for clinical or diagnostic use?

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