Making the RAW264 Whole Cell Lysate The cell pellet was vigorously resuspended in RIPA buffer (10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 1% Triton X 100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, 1 mM PMSF).

Making the RAW264 Whole Cell Lysate The cell pellet was vigorously resuspended in RIPA buffer (10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 1% Triton X 100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, 1 mM PMSF). The crude cell lysate was boiled for 2 minutes and centrifuged at 14,000 g for 10 minutes at room temperature. Cell lysate supernatant was retained and the protein concentration of the clarified cell lysate was determined using the BCA assay. The whole cell lysate concentration was diluted to 2 mg/ml, aliquoted, and archived at -20°C until required for SDS-PAGE fractionation. Table 2: Experimental treatment groups for the RAW264.7 macrophage cell activation. Tube Treatment Time RAW264.7 cells Poly IC RNA LPS Control Poly IC RNA (DAMP) LPS (PAMP) Poly IC RNA & LPS Control Poly IC RNA (DAMP) LPS (PAMP) Poly IC RNA & LPS 15 min 15 min 15 min 4 hours 4 hours 4 hours

The Correct Answer and Explanation is:

Correct Answer:

The RAW264.7 macrophage cells were divided into six experimental treatment groups based on the stimulus and treatment duration. These were:

1. Control (15 min)
2. Poly IC RNA (DAMP) – 15 min
3. LPS (PAMP) – 15 min
4. Control (4 hours)
5. Poly IC RNA (DAMP) – 4 hours
6. LPS (PAMP) – 4 hours
7. Poly IC RNA & LPS (DAMP + PAMP) – 15 min
8. Poly IC RNA & LPS (DAMP + PAMP) – 4 hours

Explanation

In this experimental design, RAW264.7 macrophage cells were treated with different immune stimuli to observe how these cells respond over time. The stimuli used include Poly IC RNA, which mimics double-stranded RNA typically associated with viral infections and acts as a Damage-Associated Molecular Pattern (DAMP), and Lipopolysaccharide (LPS), a bacterial cell wall component that serves as a Pathogen-Associated Molecular Pattern (PAMP). These molecules are known to activate innate immune receptors like TLR3 (for Poly IC) and TLR4 (for LPS), triggering inflammatory signaling pathways.

The treatments were administered for two durations: a short-term exposure (15 minutes) and a longer-term one (4 hours), allowing for the assessment of both immediate and sustained cellular responses. Including combination treatments (Poly IC RNA & LPS) allows researchers to study the potential synergistic or antagonistic effects when cells are exposed to both viral and bacterial mimics simultaneously.

Control groups for both time points are necessary to establish a baseline for comparison. The use of whole cell lysates prepared using RIPA buffer and further clarified ensures that a representative pool of intracellular proteins is available for analysis. Protein quantification via the BCA assay enables normalization across samples before SDS-PAGE and downstream analyses, such as Western blotting.

This setup is crucial for investigating the molecular mechanisms of immune activation, cytokine production, and signal transduction in macrophages under different immune challenge conditions. It can help in understanding how innate immune responses differ between DAMPs and PAMPs and how their interactions might influence inflammation or immune regulation.