RFP Expressing C57BL/6 Mouse Primary Bladder Smooth Muscle Cells

Description

When fluorescence-based studies require primary bladder smooth muscle cells that can be visually distinguished during culture, introducing a reporter after cell isolation adds another preparation step and may complicate experimental planning. Researchers also need clear information about cell identity, cryopreservation passage, culture conditions, and post-arrival storage before selecting a primary cell product.

RFP-Expressing C57BL/6 Mouse Primary Bladder Smooth Muscle Cells are isolated from the bladder tissues of pathogen-free laboratory C57BL/6 mice. The cells are grown in gelatin pre-coated tissue culture flasks with Cell Biologics' Complete Growth Medium and supplied as cryopreserved primary cells.

RFP-expressing cells are developed through transduction with RFP-lentiviral particles. Red fluorescent protein produces bright red fluorescence when exposed to yellow-green light, supporting fluorescence-based visualization of the cultured cells.

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

The cells test negative for bacteria, yeast, fungi, and mycoplasma. Under the specified primary cell 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

Features

• Primary bladder smooth muscle cells isolated from pathogen-free laboratory C57BL/6 mice
• RFP expression introduced through transduction with RFP-lentiviral particles
• Supplied as frozen cells harvested at passages 2–3
• Contains 0.5 × 10⁶ cells per mL
• Characterized by α-smooth muscle actin immunofluorescence staining using antibody A2547 from Sigma
• Tested negative for bacteria, yeast, fungi, and mycoplasma
• Recommended for expansion over 3–5 passages at a 1:2 split ratio under specified conditions
• Maintained as an adherent culture on gelatin-coated culture vessels
• Shipped frozen on dry ice and transferred to liquid nitrogen upon arrival

Applications

These RFP-expressing mouse primary bladder smooth muscle cells provide a fluorescence-visible primary cell model for research workflows requiring bladder-derived smooth muscle cells from the C57BL/6 mouse strain.

• RFP-based visualization of cultured mouse bladder smooth muscle cells
• Fluorescence-based observation of primary cell morphology and culture growth
• Reporter-expressing primary cell tracking experiments
• Primary mouse bladder smooth muscle cell culture studies
• Comparative studies involving C57BL/6 mouse primary cells
• Cell-based assays, staining, and flow cytometry workflows performed under the specified culture protocol
• Experiments requiring α-smooth muscle actin-characterized primary smooth muscle cells

Specific experimental suitability should be determined by the researcher based on the study design and applicable protocols.

Recommended Culture Reagents

Primary Cell Culture Workflow

Culture Preparation

• Perform all cell culture procedures in a biosafety cabinet using aseptic techniques.
• Sterile filter applicable media, supplements, and reagents through a 0.2 μm filter.
• Prewarm only the volume 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 frozen cells, changing medium, or splitting cells.
• Avoid repeated warming of complete culture medium.

Gelatin Coating

• Coat sterile culture flasks or dishes 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 small amount of ice remains.
2. 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 Cell Biologics cell 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 Biologics 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 the following day and every 24 hours thereafter, following the current culture 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 or magnesium.
2. Incubate with prewarmed 0.25% Trypsin-EDTA solution (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 cells detach, add 5–10 mL of complete culture medium containing FBS to neutralize the trypsin.
5. Gently pipette the cell suspension up and down several times.
6. Seed cells into fresh gelatin-coated flasks or plates at the recommended 1:2 split ratio.
7. Return the culture vessels to a humidified 37°C, 5% CO₂ incubator.
8. Change the medium the following day and every 24 hours thereafter.

Preparation for Experiments

• Once cells reach confluence, they may be allowed to overgrow for up to 24 hours before cell-based assays, staining, flow cytometry, or other designed experiments.
• Change the medium daily and pre-wash the cells twice with 1X PBS between medium changes.
• Before an experiment, cells may be passed through a 40 μm cell strainer to remove cell clumps and large dead or floating cells.

Laboratory Cryopreservation

• Add 1 mL of CB6916 Freezing Buffer to each vial and quickly resuspend the cell pellet.
• Place the vials in a Nalgene "Mr. Frosty" freezing container filled with 100% isopropanol at −80°C for 6–12 hours.
• Transfer the vials to a liquid nitrogen tank for long-term storage.

The temporary −80°C step applies only to controlled laboratory cryopreservation. Received cryopreserved cells should be transferred from dry ice directly to liquid nitrogen and should not be kept at −80°C for routine storage.

Quality Control

Shipping, Storage and Handling

• Cryopreserved cells are shipped frozen on dry ice by overnight service.
• Upon arrival, transfer the frozen vial immediately to a liquid nitrogen storage tank until ready for use.
• Do not keep received cryopreserved primary cells at −20°C or −80°C for routine storage.
• Avoid repeated freezing and thawing.
• Handle animal cells with appropriate caution because no testing procedure can guarantee the complete absence of infectious agents.
• Use aseptic cell culture procedures in an appropriate biosafety cabinet.

Documents

Primary Cell Thawing & Culture Protocol

Archived Primary Cell Culture Protocol

Cell Culture Products Safety Data Sheet

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

Frequently Asked Questions

What are RFP-Expressing C57BL/6 Mouse Primary Bladder Smooth Muscle Cells?

They are primary smooth muscle cells isolated from bladder tissues of pathogen-free laboratory C57BL/6 mice. RFP expression is introduced through transduction with RFP-lentiviral particles, and the cells are supplied cryopreserved.

Are these primary or immortalized mouse bladder smooth muscle cells?

These are primary mouse bladder smooth muscle cells. They are not described as immortalized cells.

How is RFP introduced into the primary cells?

RFP-expressing cells are developed through transduction with RFP-lentiviral particles. Red fluorescent protein produces bright red fluorescence when exposed to yellow-green light.

How many cells are supplied in each vial?

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

At what passage are the cells cryopreserved?

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

How many passages can the cells be expanded?

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

How are the mouse bladder smooth muscle cells characterized?

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

What quality control testing is documented?

The cells are documented as negative for bacteria, yeast, fungi, and mycoplasma.

What medium is recommended for these primary bladder smooth muscle cells?

The suggested medium is M2268 Complete Smooth Muscle Cell Medium w/ Kit – 500 mL.

Do culture flasks need to be coated before seeding the cells?

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

What incubation conditions are recommended?

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

Can the received cells be stored in a −80°C freezer?

No. Transfer the received cryopreserved cells from the dry ice shipping container to liquid nitrogen immediately upon arrival. The −80°C step in the freezing protocol is only a temporary controlled-freezing step before transfer to liquid nitrogen.

Are these cells intended for clinical or therapeutic use?

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