Introduction:

Embarking on your first laboratory experiments can be both exciting and overwhelming. You can use this guide to ensure your experiments are well-planned and the planning process runs smoothly. We will use an example to explain how to plan experiments from the beginning. So let’s dive in and uncover the steps to successfully design your first laboratory experiments.

Step 1: Analyze the Research Question

Begin by clearly defining your research question. For instance, “Do macrophage-fibroblast interactions influence the metastatic spread of tumor cells?”

Step 2: Exclude Keywords from the Research Question

Identify the keywords in your research question and extract them. In our example, the extracted keywords are “interaction” and “metastatic spread.”

Step 3: Determine Descriptive Characteristics

Think about what characteristics could describe the keywords you excluded. In this case:

– As a result of interaction, things overlap, but they don’t exist separately in the same way. Thus, this cooperation can diminish, amplify, or even generate new effects.

– The metastatic spread has several amplified characteristics, depending on its stage. There is increased proliferation, angiogenesis, transcription of proliferative genes, and decreased transcription of dormant genes.

Which assays can you use to measure separate characteristics of the keywords?

Proliferation	CCK-8, BrDU
Gene Expression	PCR
Extracellular Proteins (secreted)	ELISA
Intracellular Proteins and their distribution	IF
Intracellular protein	WB

Step 4: Explore Existing Literature to Shape Expectations

Research what is known in the literature about your research question to shape your expectations. In our case, macrophages and fibroblasts should interact to promote metastasis.

Step 5: Define the Desired Outcome

Decide what effect you want to observe in your experiments. This will further refine your focus and guide your experimental design. In our case, the desired outcome is a change in the metastatic spread.

Step 6: Determine the Cause

Consider how you will prove the cause of the observed effect. In our case, we know that metastatic cells spread due to the interaction between macrophages and fibroblasts. We wonder if this interaction occurs directly with tumor cells or indirectly through soluble messengers. We are not interested in tumor cell-macrophage or tumor cell-fibroblast interactions. Thus, we will focus on an indirect approach that prevents other combinations of cell-cell interactions.

We will test this through different experimental setups.

Experimental Design:

Part 1: Examining the Effect of Interaction on Tumor Cells. Test tumor cells with and without supernatant derived from macrophages grown with fibroblasts. Identify if the change in the metastatic spread is present.

Part 2: Identifying the Source of the Effect. Identify whether the effect is caused by macrophages, fibroblasts, or their interaction. Investigate what drives the impact.

– Group 1: Tumor cells treated with supernatant from macrophages grown with fibroblasts.
– Group 2: Tumor cells treated with supernatant from macrophages alone.
– Group 3: Tumor cells treated with supernatant from fibroblasts alone.

Optimizing Experimental Conditions:

To ensure reliable results, consider the following factors:

1. Type of media and its volume. Choose a medium type and volume that supports cell growth and viability during the experiment.

2. Decide on Main Control. Part 1: Tumor cells without supernatant. Part 2: Tumor cells treated with supernatant from macrophages grown with fibroblasts.

3. Number of cells and incubation time. Ensure that the cell density allows proliferation without overcrowding. Adjust the number of cells and the incubation time accordingly. Some genes and secreted proteins may need longer incubation times. Follow color changes in the cells’ media throughout the experiment, as they could show overcrowding.