GFP Expressing C57BL/6 Mouse Primary Stomach Smooth Muscle Cells

Description

GFP-Expressing Mouse Primary Stomach Smooth Muscle Cells from Cell Biologics are isolated from the stomach tissues of pathogen-free laboratory C57BL/6 mice and grown in gelatin pre-coated tissue culture flasks with Cell Biologics' Complete Growth Medium.

GFP-expressing cells are developed through transducing GFP-lentiviral particles. The green fluorescent protein (GFP) gives rise to bright green fluorescence when exposed to blue light.

Cells at passages 2–3 are harvested from flasks and cryopreserved in vials. Each vial contains 0.5 × 10⁶ cells per mL and is delivered frozen. GFP-Expressing Mouse Primary Stomach Smooth Muscle Cells are characterized by immunofluorescence staining with an α-smooth muscle actin antibody (A2547, Sigma).

These cells are negative for bacteria, yeast, fungi, and mycoplasma, and can be expanded for 3–5 passages at a split ratio of 1:2 under the specified cell culture conditions.

Specifications

Features

• Isolated from stomach tissues of pathogen-free C57BL/6 mice
• GFP expression introduced through transducing GFP-lentiviral particles
• Harvested and cryopreserved at passages 2–3
• Contains 0.5 × 10⁶ cells per mL per vial
• Characterized by α-smooth muscle actin immunofluorescence staining (A2547, Sigma)
• Tested negative for bacteria, yeast, fungi, and mycoplasma
• Can be expanded for 3–5 passages at a split ratio of 1:2 under specified conditions
• Cultured on Gelatin-Based Coating Solution (Catalog No. CB6950)
• Shipped frozen on dry ice overnight; stored in liquid nitrogen upon arrival

Cell Culture Workflow

Culture Preparation

• Perform all cell culture procedures in a biosafety cabinet.
• Use aseptic techniques to prevent microbial contamination.
• Sterile filter all media, supplements, and reagents through a 0.2 μm filter.
• Prewarm only the amount of cell culture medium needed right away (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 warming of complete culture medium is not recommended.

Gelatin Coating of Flasks or Dishes

• Coat sterile culture dishes or flasks with Gelatin-Based Coating Solution (Catalog No. CB6950) for 2 minutes.
• Aspirate excess solution before seeding cells.

Recovery of Cryopreserved Cells

1. Thaw the vial rapidly in a 37°C water bath for less than 1 minute or until only a few ice pieces remain.
2. Disinfect the exterior of the vial with 70% ethanol.
3. Transfer cells into a sterile 15 mL conical tube containing 5 mL of prewarmed complete culture medium.
4. Rinse the vial with 0.5–1 mL of medium to recover remaining cells.
5. Centrifuge cells at 200 × g for 5 minutes.
6. Carefully remove supernatant with a sterile Pasteur pipette.
7. Resuspend the cell pellet in 5 mL of prewarmed complete medium.
8. Seed cells into a gelatin-coated T25 flask.
9. Incubate in a 37°C, 5% CO₂ incubator and leave the culture undisturbed for 12–16 hours.
10. Change the medium the next day and every 24–48 hours thereafter.
11. Check cell confluence and morphology daily under a microscope.

Primary Cell Expansion (Subculture)

• Wash adherent cells twice with 1X PBS (calcium- and magnesium-free).
• Incubate cells with prewarmed (37°C) 0.25% Trypsin-EDTA solution (Catalog No. CB6914) for 3–5 minutes.
• Use 2 mL of Trypsin-EDTA solution for a T25 flask and 3.0 mL for a T75 flask.
• As soon as cells detach, add 5 mL of Complete Growth Medium to neutralize trypsin, and gently pipette up and down 1–2 times.
• Transfer cell suspension to a centrifuge tube and centrifuge at 200 × g for 5 minutes.
• Carefully remove supernatant and resuspend cells in fresh medium.
• Seed cells into fresh gelatin-coated culture vessels at a split ratio of 1:2.

Preparation for Experiments

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

Cryopreservation

• Add 1 mL of Freezing Buffer (Catalog No. CB6916 / Freezing Media) per vial and quickly resuspend the cell pellet.
• Place vials in a Nalgene "Mr. Frosty" freezing container filled with 100% isopropanol at -80°C for 6–12 hours.
• Transfer vials to a liquid nitrogen tank for long-term storage.

Quality Control

Storage and Handling

• Cryopreserved cells are shipped frozen with dry ice overnight.
• Upon arrival, transfer frozen cells immediately to a liquid nitrogen storage tank (-180°C) until ready for use.
• Primary cells can never be kept in a -20°C or -80°C freezer for routine storage.
• Repeated freezing and thawing of cells are not recommended.
• Investigators should handle cells with caution and treat all animal cells as potential pathogens.
• For research use only. Not authorized for human use, in vitro diagnostic, or therapeutic procedures.

Documents

C57-6087GFP Product Datasheet

Primary Cell Culture Protocol

Primary Cell Culture Protocol (Supplementary)

Cell Culture Products Safety Data Sheet

Frequently Asked Questions