Question

With the large pipette labeled "PBS," add 5 drops of PBS (or, \(200 \mu \mathrm{L}\) with a digital pipettor) to each well, beginning with Row A and finishing with Row H. Don't overfill the wells and allow solutions from different wells to mix. This wash step is a bath not a shower-add the PBS gently! After all wells have been filled, use the small pipette labeled "Ag1" to gently remove the PBS from wells in Rows A through D. Repeat with the small pipette labeled "Ag2" for Rows E through H. Dispose of the PBS in the disposal beaker.

Short answer

Add 5 drops of PBS to each well, then use 'Ag1' pipette to remove PBS from Rows A-D, and 'Ag2' pipette for Rows E-H.

Step-by-step solution

- Prepare Pipettes

Ensure that the large pipette labeled 'PBS' is ready for use. If using a digital pipettor, set it to dispense 200 \(\mu\mathrm{L}\).

- Dispense PBS

Add 5 drops of PBS (or 200 \(\mu\text{L}\)) to each well starting from Row A and ending with Row H. Do not overfill the wells and ensure not to mix the solutions from different wells.

- Remove PBS from Rows A to D

Using the small pipette labeled 'Ag1', gently remove the PBS from each well in Rows A through D. Dispose of the removed PBS in the disposal beaker.

- Remove PBS from Rows E to H

Using the small pipette labeled 'Ag2', gently remove the PBS from each well in Rows E through H. Dispose of the removed PBS in the disposal beaker.

Key concepts

Pipette Use

Pipettes are essential tools in laboratory settings, used to measure and transfer small volumes of liquid precisely. In this exercise, two types of pipettes are employed: a large one for adding PBS (phosphate-buffered saline) and smaller ones for removing it. When working with pipettes, always ensure that they are correctly calibrated to avoid inaccurate measurements. For disposable pipette tips, remember to change tips between uses to prevent cross-contamination.
To use a pipette, follow these steps:

• Set the desired volume correctly (for digital pipettors).
• Attach an appropriate pipette tip.
• Press the plunger to the first stop before inserting it into the liquid.
• Release the plunger gently to draw the liquid into the tip.
• Dispense the liquid slowly and steadily by pressing the plunger to the second stop.

Ensuring proper pipetting technique is crucial for obtaining reliable and reproducible results in any laboratory procedure.

PBS Solution

PBS, or phosphate-buffered saline, is a buffer solution commonly used in biological research. It maintains a consistent pH and osmolarity, which makes it ideal for washing cells in culture, maintaining cell viability, and serving as a diluent for reagents. In this exercise, PBS is used to wash the wells of the plate.
The components of PBS are:

• NaCl (sodium chloride)
• KCl (potassium chloride)
• Na₂HPO₄ (sodium phosphate dibasic)
• KH₂PO₄ (potassium phosphate monobasic)

When preparing PBS, ensure that the solution is sterile to prevent contamination. Using PBS correctly helps in minimizing stress on cells and maintains the integrity of experimental conditions.

Laboratory Technique

Mastering laboratory techniques is fundamental for conducting experiments accurately and safely. This exercise highlights several important techniques, including the gentle addition of liquids and preventing cross-contamination. Pouring PBS too quickly can disrupt cell layers or mix solutions from different wells, leading to imprecise results.
Key points to remember include:

• Handle pipettes and reagents gently.
• Avoid touching the inside of pipette tips or wells.
• Discard any remaining liquid from single-use pipette tips appropriately.
• Ensure all equipment is clean and, if necessary, sterilized before use.

Practicing good laboratory techniques ensures not only the reliability of your data but also the safety of the laboratory environment.

Well Plate Handling

Well plates, or microplates, are used for various assays in which small volumes of liquid are tested across multiple wells. In this exercise, you are required to handle a well plate meticulously to avoid contamination and ensure accurate results.
Some tips for effective well plate handling are:

• Hold the plate steady to avoid spillage.
• Label each row and column for easy identification.
• Never fill the wells to the brim to prevent overflow and cross-well contamination.
• When adding or removing liquids, always use a consistent amount to maintain uniformity across experiments.

Following these guidelines will help maintain the integrity of your well plate assays, leading to more reliable and interpretable data.