How do I set up the LeviCell system for live cell enrichment?

This getting started guide will provide you with a step by step procedure for live cell enrichment

There are two steps to running a live cell enrichment experiment:

• Prepare Single Cell Suspension
• Run the LeviCell instrument

Prepare Single Cell Suspension

To run the LeviCell instrument, you'll need an input of 220 µl. The number of cells you choose to load will vary based on your specific sample. For your initial sample run, it's recommended to use a final concentration of 150 mM Levitation Agent, which can be adjusted as necessary.

To prepare the single cell suspension, you can either:

1. Add the Levitation Agent directly to a stock solution of cells, or
2. Resuspend the cells in Levitation Buffer after pelleting the required number of cells 

Adding Levitation Agent directly to a stock solution

When working with a stock solution of cells, you have the option to add the Levitation Agent directly to your sample. This is a convenient method that saves time and effort. First it is important to QC your stock solution and get an estimate number of cells in the total stock. From here, you can take the desired input of stock solution and mix it with the required Levitation Agent and buffer to create a total of 250 µl of cell suspension. This suspension can then be taken to the LeviCell instrument for live cell enrichment experimentation.

It's important to note that the amount of buffer added should be adjusted accordingly if you are using a different volume of cell stock. When adding the Levitation Agent directly to the stock solution, ensure that it is mixed thoroughly to achieve a homogenous suspension. This will help to ensure that you get accurate and consistent results when running the LeviCell instrument. In the table below, an example is given where 20 µl of stok solution is used.

ii) Resuspending cells in Levitation Buffer

When resuspending cell pellets:

1. Prepare Levitation Buffer in advance
2. Pellet the required cell concentration
3. Resuspend the pellet in Levitation buffer

Prepare Levitation Buffer

 In new 1.5 mL tube, prepare Levitation Buffer as follows

The above mixture will provide with a 150 mmol Levitation Buffer. Vortex  the mixture well to completely mix the Levitation Buffer.

Pellet the required cell concentration

To pellet the required amount of cells, you can use a centrifuge at 300 relative centrifugal force (RCF) for 5 minutes. After centrifugation, carefully remove the supernatant without disturbing the cell pellet. You may now use the Levitation Buffer you prepared in advance to resuspend the cell pellet. It is important to ensure that the cell pellet is completely resuspended in the Levitation Buffer to achieve accurate and consistent results. A thorough mixing of the Levitation Buffer and cell pellet can be achieved by pipetting up and down gently.

Run LeviCell Instrument

The Experiment Manager user interface lets you start a run. From the user interface, select the protocol which matches your desired levitation time:

1. When ready to start run, mix sample thoroughly by pipetting up and down gently 5X and immediately load 220 μL into input well.
2. The default split line is set at “0”. When levitation is finished, the split line setting can be adjusted as necessary (from -15 to +15) to better separate live cell band from dead cells/ debris.
3. Harvest your cells from Top well output into a 1.5 mL low-bind tube. Ensure all liquid from the output well and serpentine channel is collected.
4. Measure the final output volume using a pipette. When split line is set to 0, typical recovery is between 70-100 μL.

Set aside 15 μL for cell counting.

Count Cells

After collecting 15 μL aliquots of both the input and output from the LeviCell instrument, it's important to determine the number of cells present in your sample. To accurately count the cells, it's recommended to use a Dual Stain cell counter along with a live/dead stain like AO/PI. This type of stain allows you to distinguish between live and dead cells, giving you a more accurate count of viable cells in your sample.

To use a Dual Stain cell counter, first dilute your sample in a suitable buffer and then mix it with the staining solution. After incubating for a few minutes, load the sample onto the cell counter and follow the manufacturer's instructions to analyze the cells. The machine will detect live cells as green and dead cells as red, allowing you to count the number of viable cells in your sample accurately.

By counting the cells present in both the input and output samples, you can calculate the yield and viability increase. This will help you to assess the success of your live cell enrichment experiment and make any necessary adjustments for future experiments.