GFP Expressing C57BL/6 Mouse Primary Pulmonary Vein Fibroblasts

Description

Experiments involving pulmonary vascular fibroblasts require a cell source with clearly defined tissue origin, strain background, fluorescence characteristics, and culture conditions. Using general lung fibroblasts in place of pulmonary vein-derived cells may not provide the tissue specificity required for studies focused on the pulmonary venous environment.

GFP-Expressing C57BL/6 Mouse Primary Pulmonary Vein Fibroblasts are isolated from the pulmonary vein tissue of pathogen-free C57BL/6-Tg(CAG-EGFP)1Osb/J laboratory mice. The cells provide a GFP-expressing primary fibroblast model for fluorescence-based observation and standard cell culture research involving mouse pulmonary vein-derived fibroblasts.

Cells are cultured in gelatin-coated tissue culture vessels with complete fibroblast growth medium, expanded, and cryopreserved in vials for frozen delivery. Each vial contains at least 1 × 10⁶ cells per mL.

Cell identity is characterized by immunofluorescence staining with an anti-FSP1/S100A4 antibody. The cells are tested negative for bacteria, yeast, fungi, and mycoplasma. Under the specified culture conditions, they can be expanded for 3–5 passages using a recommended split ratio of 1:2.

GFP-expressing C57BL/6 mouse primary pulmonary vein fibroblasts are supplied for research use only. They are not authorized for human use, in vitro diagnostic procedures, or therapeutic applications.

Specifications

Features

• Primary fibroblasts derived specifically from C57BL/6-GFP mouse pulmonary vein tissue
• GFP-expressing cells suitable for fluorescence-based observation in cell culture workflows
• Cryopreserved format for controlled recovery and experimental scheduling
• At least 1 × 10⁶ cells per mL
• Characterized by anti-FSP1/S100A4 immunofluorescence staining
• Tested negative for bacteria, yeast, fungi, and mycoplasma
• Expandable for 3–5 passages under the specified culture conditions
• Recommended 1:2 split ratio for primary cell expansion
• Supported by defined thawing, culture, coating, and cryopreservation protocols

Applications

These GFP-expressing mouse primary pulmonary vein fibroblasts provide a tissue-specific primary cell model for in vitro studies requiring pulmonary vein-derived fibroblasts and an intrinsic fluorescent reporter. Applications should be selected according to the laboratory’s experimental design and the characteristics of primary cells.

• Fluorescence imaging: Observation of GFP-expressing primary fibroblasts in culture without introducing an additional fluorescent label for routine visualization.
• Immunofluorescence studies: Evaluation of cultured pulmonary vein fibroblasts using fluorescence-based staining and imaging workflows.
• Flow cytometry: Analysis of GFP-expressing cells and additional fluorescent markers using appropriate compensation and gating controls.
• RT-PCR: Gene-expression studies using RNA isolated from cultured C57BL/6 mouse pulmonary vein fibroblasts.
• Western blotting: Protein-expression analysis following culture under defined experimental conditions.
• Immunoprecipitation: Investigation of proteins and protein interactions in pulmonary vein-derived fibroblast cultures.
• Cell derivative generation: Preparation of cultured cell derivatives for downstream research applications.
• Pulmonary vascular fibroblast research: In vitro studies requiring primary fibroblasts derived specifically from mouse pulmonary vein tissue.

Application suitability depends on the experimental design. No disease-specific, diagnostic, therapeutic, or clinical performance claim is made.

Quality Control

Cell Culture Workflow

Culture Preparation

• Perform all 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 volume of medium required for immediate use to 37°C.
• Use approximately 5 mL of medium for a T25 flask and 8–10 mL for a T75 flask when recovering cells, changing medium, or splitting cells.
• Avoid repeatedly warming complete culture medium.

Gelatin Coating

1. Add CB6950 Gelatin-Based Coating Solution to a sterile culture flask or dish.
2. Allow the 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 the exterior with 70% ethanol.
3. Transfer the cell suspension into a sterile 15 mL conical tube containing 5 mL of prewarmed complete 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 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. Incubate the flask in a humidified incubator at 37°C with 5% CO₂.
9. Leave the culture undisturbed for 12–16 hours.
10. Change the medium the following day and every 24 hours thereafter.
11. Check cell morphology and confluence 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 prewarmed 0.25% Trypsin-EDTA Solution (CB6914).
3. Use approximately 0.5–1 mL for a T25 flask or 2 mL for a T75 flask.
4. Incubate for 3–5 minutes and monitor cell detachment.
5. Once cells detach, add 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 at a recommended split ratio of 1:2.
8. Return the culture vessels to a humidified 37°C, 5% CO₂ incubator.

Cryopreservation

1. Harvest the cells using the specified trypsinization procedure.
2. Centrifuge the cell suspension at 200 × g for 5 minutes.
3. Remove the supernatant and add 1 mL of CB6916 Freezing Medium per cryovial.
4. Place the cryovials in a controlled-rate freezing container with 100% isopropanol.
5. Freeze at −80°C for 6–12 hours as an intermediate controlled-freezing step.
6. 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 primary cells.
• The −80°C step described in the freezing protocol is only an intermediate controlled-freezing step before transfer to liquid nitrogen.
• Repeated freezing and thawing are not recommended.
• Handle animal-derived cells as potential biohazards and follow institutional biosafety procedures.
• Review the supplied Safety Data Sheet before use.

Documents

Primary Cell Thawing & Culture Protocol

Archived Primary Cell Culture Protocol

Cell Culture Products Safety Data Sheet

Use the current Primary Cell Thawing & Culture Protocol for routine handling. The archived protocol is retained as a supplementary reference and may contain earlier procedural parameters.

Frequently Asked Questions

What are GFP-expressing C57BL/6 mouse primary pulmonary vein fibroblasts?

They are primary fibroblasts isolated from the pulmonary vein tissue of pathogen-free C57BL/6-Tg(CAG-EGFP)1Osb/J laboratory mice and supplied as cryopreserved GFP-expressing cells for research use.

What is the difference between pulmonary vein fibroblasts and general mouse lung fibroblasts?

This product is derived specifically from mouse pulmonary vein tissue. General mouse lung fibroblast products may be isolated from broader lung tissue and should not be assumed to have the same tissue origin.

How many cells are supplied?

Each cryopreserved vial contains at least 1 × 10⁶ cells per mL.

How are the cells characterized?

Cell identity is characterized by immunofluorescence staining with an anti-FSP1/S100A4 antibody.

What contamination testing is documented?

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

How many passages can the cells be expanded?

The cells can be expanded for 3–5 passages under the specified culture conditions. The recommended split ratio is 1:2.

What medium is recommended for these primary fibroblasts?

M2267 Fibroblast Medium w/ Kit – 500 mL is the suggested complete medium for this product.

Do the culture vessels require coating?

Yes. Culture vessels should be coated with CB6950 Gelatin-Based Coating Solution for 2 minutes, after which the excess solution should be aspirated before cell seeding.

What culture conditions are recommended?

Culture the cells in a humidified incubator at 37°C with 5% CO₂. Check cell morphology and confluence daily.

How should the cryovial 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 with 70% ethanol before transferring the cells into prewarmed medium.

Should residual freezing medium be removed after thawing?

Yes. The current protocol recommends centrifuging the cells at 200 × g for 5 minutes after thawing to remove residual DMSO-containing freezing medium.

How should the cells be stored upon arrival?

Transfer the cryopreserved cells from the dry ice shipping container to a liquid nitrogen storage tank immediately upon arrival.

Can the cells be stored at −80°C after delivery?

No. A −80°C freezer is used only as an intermediate controlled-freezing step for 6–12 hours during cryopreservation. Delivered cryopreserved cells should be stored in liquid nitrogen.

What research applications are listed for these cells?

The cells may be used in standard cell culture research workflows including RT-PCR, Western blotting, immunoprecipitation, immunofluorescence, flow cytometry, and generation of cell derivatives for downstream research.

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 applications.