The RAW 264.7 cell line, derived from a murine macrophage, is a widely used model in immunology research. These immortalized cells provide a robust platform for studying inflammation, immune responses, and phagocytic activity. Their ability to perform functions like pinocytosis and phagocytosis makes them valuable tools for understanding host-pathogen interactions and screening anti-inflammatory compounds. This protocol details the steps necessary to successfully culture, maintain, and store this versatile cell line.
Essential Culture Components and Equipment Setup
Successful RAW 264.7 cell culture relies on a sterile environment and a specific growth medium formulation. The primary medium utilized is Dulbecco’s Modified Eagle’s Medium (DMEM). This base medium requires the addition of 10% Fetal Bovine Serum (FBS) to supply necessary growth factors and nutrients. 1% Penicillin-Streptomycin (P/S) is commonly included to prevent bacterial contamination, though some protocols may omit this antibiotic.
The physical environment must be controlled. A sterile biosafety cabinet (Level 2) is needed for all handling procedures to maintain aseptic conditions and protect the user. The cells thrive in a humidified incubator set to 37°C. The atmosphere requires a 5% carbon dioxide (\(\text{CO}_2\)) concentration, which maintains the correct pH balance of the bicarbonate-buffered DMEM.
Initiating Culture from Cryopreserved Stocks
The process of reviving cryopreserved cells must be executed rapidly to maximize cellular viability. Vials are typically stored in the vapor phase of liquid nitrogen and should be immediately transferred to a 37°C water bath for quick thawing. The vial should be gently swirled until only a small ice crystal remains, taking care to keep the cap out of the water. Since the cryoprotectant, dimethyl sulfoxide (DMSO), is cytotoxic at warmer temperatures, rapid thawing is essential.
Once thawed, the exterior of the vial must be decontaminated with 70% ethanol before transfer into a sterile hood. The cell suspension is then transferred to a centrifuge tube containing a large volume of pre-warmed complete growth medium, which dilutes the toxic DMSO. The tube is centrifuged at a low speed to pellet the cells. After centrifugation, the supernatant containing the DMSO is carefully discarded, and the cell pellet is resuspended in fresh medium for initial seeding into a culture vessel.
Routine Subculturing and Cell Counting
For ongoing maintenance, RAW 264.7 cells are typically subcultured when they reach 70% to 80% confluency, although some protocols suggest a maximum of 60% to 75%. Macrophages generally do not tolerate high density well, and allowing the culture to become overly confluent can lead to slower growth and potential changes in cell characteristics. Unlike many adherent cell lines, RAW 264.7 cells adhere loosely to the culture surface, meaning they should not be treated with harsh enzymatic solutions like trypsin.
The cells are instead detached mechanically, most commonly by using a cell scraper to gently dislodge them from the flask surface. Alternatively, gentle aspiration and expulsion of medium directly onto the cell monolayer can be used for detachment. The resulting cell suspension is collected into a centrifuge tube and mixed to break up any cell clumps. A small aliquot of this suspension is then mixed with Trypan Blue dye to assess viability for cell counting.
Live cells will exclude the Trypan Blue dye, while dead cells will take it up, appearing blue under a microscope. The total number of viable cells is counted using a hemocytometer, allowing for the calculation of the cell concentration. Based on the cell count, the suspension is diluted into fresh culture medium at a subcultivation ratio to seed new flasks at the optimal density for continued growth. The recommended seeding density is often between \(5.0 \times 10^4\) and \(7.5 \times 10^4\) viable cells per \(\text{cm}^2\) of surface area.
Long-Term Storage (Cryopreservation)
Cryopreservation is a process used to halt the cell’s metabolic activity, preventing genetic drift. The cells must be in a healthy, actively growing state before freezing. The optimal cell concentration for freezing is generally between \(5 \times 10^6\) and \(1 \times 10^7\) cells per milliliter of freezing medium. The freezing medium is prepared by mixing a high-quality serum, such as FBS, with a cryoprotective agent, typically 90% FBS and 10% DMSO.
DMSO penetrates the cell membrane, lowering the freezing point and preventing the formation of damaging intracellular ice crystals. This mixture should be added drop-wise to the concentrated cell pellet while the cells are kept on ice to minimize DMSO toxicity. Once dispensed into labeled cryovials, the cells must be frozen at a slow, controlled cooling rate, ideally one degree Celsius per minute, to maximize post-thaw viability. This controlled rate is achieved using a specialized device, such as a Mr. Frosty container, placed into a \(-80^\circ\text{C}\) freezer overnight. For long-term storage, the cryovials are then transferred to the vapor phase of a liquid nitrogen tank, where they can remain viable for many years.
Common Contamination and Morphological Issues
When observed under a phase-contrast microscope, healthy, non-activated RAW 264.7 cells appear round or oval and are phase-bright. They are adherent, but they can sometimes appear semi-adherent, with some cells floating in the medium, especially when ready for subculture. The appearance of elongated or spindle-shaped cells can indicate a polarized or activated state, which may be a response to stimuli in the medium or a sign of differentiation.
Contamination is a frequent challenge in cell culture, and visual inspection is the first line of defense. Bacterial contamination often presents as small, rapidly moving particles between the cells, which can make the medium appear turbid or cloudy. Fungal contamination is recognizable by filamentous structures or budding yeast, which are larger than bacteria and often grow in distinct clusters. Mycoplasma contamination is not visible by standard light microscopy and requires specific DNA-staining or PCR-based assays for detection. If contamination is suspected, the affected culture should be immediately discarded, and the incubator and hood should be thoroughly disinfected to prevent spread.