RAW 264.7 is a mouse macrophage cell line originally isolated by the National Cancer Institute (NCI) in the 1970s, derived from tumor tissue induced by Abelson murine leukemia virus. These cells are round or spindle-shaped (spindle shape indicates a polarized state) and adhere to surfaces. Under certain conditions, they exhibit pseudopod growth. Due to their phagocytic and endocytic properties, they are widely used in research as a model for inflammatory cells, including studies on immune responses, inflammation, infectious diseases, drug screening, and toxicity evaluation.

Cell Morphology

RAW 264.7 cells are round or spindle-shaped (spindle shape indicates polarization) and are adherent. Under normal culture conditions, the cells are round or oval, and the ideal state is small and translucent. Sometimes, cells loosely adhere, cluster, and round up, which is normal. However, if more than 10% of the cells become spindle-shaped (indicating polarization), caution is needed.

Culture Conditions

• Medium: DMEM + 10% FBS + 1% PS (recommended media: 1640, high-glucose media, and high-quality serum).
• Incubation: 95% air + 5% CO₂ at 37°C.

Challenges in Culturing RAW 264.7 Macrophages

When starting the culture, common issues include cell differentiation (most common), pseudopod growth, difficulty in digestion, slow growth, and floating without adhering.

Experimental Procedure

Materials

• Cell line: Mouse monocyte macrophage cell line RAW264.7
• Reagents: High-glucose medium, PBS, serum-free cell cryopreservation solution

Techniques

Cell Resuscitation: Rapidly thaw a cryovial containing 1 mL of RAW264.7 cell suspension in a 37°C water bath (generally for 1 minute), shaking it to ensure even thawing (be careful not to immerse the vial opening in the water to avoid contamination). Add 4 mL of pre-warmed medium and mix well. Centrifuge at 1000 rpm for 3 minutes, discard the supernatant, add 1–2 mL of medium, and gently pipette to resuspend the cells. Transfer the cell suspension into a flask with fresh medium for overnight incubation (T25 flasks are recommended for initial culturing; once the cells are in good condition, transfer them to larger dishes).

Tips:

• RAW264.7 cells tend to polarize, so it’s important to warm the medium during resuscitation to prevent polarization.
• Evaluate the need for a medium change the next day based on cell condition. Frequent changes may disrupt cell growth.
• RAW264.7 cells prefer high-density growth. If the cell density is too low, growth will be slow.

Cell Passage: transfer (or subculture) your cells to a new dish when they reach 80-90% confluence. If the density is too low, they will grow slowly and polarize easily. Discard the culture medium, and if the cells appear unhealthy, rinse the RAW264.7 cells once with 1 mL of medium. Add 2 mL of medium to cover all the cells, then gently pipette to detach them. Collect the detached cells and suspension into a sterile centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend in 2–3 mL of medium. Transfer or subculture the cells at a ratio of 1:2 or 1:3 into new flasks and mix evenly.

Tips:

• Since RAW264.7 cells polarize easily, it’s not recommended to use trypsin for digestion or PBS for pipetting during passage.
• If polarization occurs, lightly pipette the cells and replace the dish. If polarization persists, control the polarization rate by adjusting the cell density and passage schedule.
• If cells become spindle-shaped or exhibit pseudopods in large numbers during normal culture, they should not be used, as they may lead to abnormal data.

Cell Cryopreservation: First, add 1 mL of medium to gently mix and discard any unhealthy cells. Collect the cells using PBS, centrifuge at 1000 rpm for 45 minutes, discard the supernatant, wash with PBS, and discard the PBS. Count the cells, adjust to a density of 5 × 10⁶ to 1 × 10⁷ cells/mL, and add serum-free cryopreservation solution. Aliquot 1 mL of the suspension into each cryovial, label the generation and date, and store at -80°C. (Labs with ample resources may choose to produce large batches and freeze them to reduce batch variability).

Common Issues

– What if many RAW264.7 cells have polarized during culture?

If the polarization rate is high, it’s recommended to discard the culture and start again. However, a small amount of differentiation is normal and generally does not affect experimental results.

– Slow growth or frequent polarization in 1640 medium?

Try using a higher glucose concentration in the medium or increase the FBS concentration. If the issue persists, consider possible contamination (e.g., mycoplasma).

– Cells do not adhere after passage?

If cells do not adhere, centrifuge them again, resuspend in fresh medium, and ensure proper pipetting to avoid cell clumping.

– Timing for passage or medium change?

Generally, passage the cells every 1–2 days, but do not exceed 3 days, as overgrowth may lead to cell death or polarization.