Sample requirements
- Optimal results using viable cells (viability over 70%) and isolated nuclei
- Please get in touch with our team if cell viability is below 70%
- Single cells or nuclei are sorted in cell capture plates containing cell buffer.
- Cell capture plates will be provided by Single Cell Discoveries unless otherwise specified
- Labeling: label plates clearly on the seal and sides, making sure the labels match the information in the sample submission file
- Alternatively, single cells of interest can be sorted and cryopreserved before shipment to SCD. Make sure our team receives thawing instructions to minimize cell loss
- Cell numbers: >10^6 cells per sample, minimal 500.000 cells per sample
- Labeling: label tubes clearly on the lids and sides, making sure the labels match the information in the sample submission file
Plate preparation
- Please read through the sorting instructions below and do not hesitate to reach out if you have any questions.
- We will provide you with an empty plate to calibrate the FACS machine
- Leave the first column of the plate empty; these wells will be used for addition of control samples on the same plate to assess the performance of the plate.
- Make sure the plates are sealed using the provided seals and the plate ID is readable.
Shipment
In a plastic bag or cardboard Eppendorf box, or when shipping plates, tape the plates together, making sure the right side is up, on dry-ice. Please check our shipping section for more information.
FACS sorting
The essentials
There are a few things that are essential for a successful sort:
- The quality of your cells
If the quality of the cells is not sufficient, the sequencing data will also be of low(er) quality. Therefore, we advise checking whether your cells survive the dissociation and the FACS sort before you plan your definitive sorting experiment. For more information on how to check the cell quality, please check our cell quality guide. - A proper gating strategy
Without proper gating, you will end up with a suboptimal or even wrong population of cells. As a minimum, we advise selecting for live cells, by using a DAPI staining or other live/dead marker. It is also advisable to sort a few cells onto a microscopy slide or culture dish to check whether the morphology of the sorted particles fits your cell type of interest. - Proper calibration of the FACS sorter
If the FACS machine is not appropriately aligned, cells will unfortunately not end up in the wells (correctly). Hence, it is essential to calibrate your FACS machine – every time – you start a sorting experiment. You can use the empty 96-well plate that was shipped with the cell capture plates for the calibration. Seal the plate using one of the plate seals and sort FACS fluid/beads onto the foil at wells e.g.: A1-3 and H11-13. Try to position the droplets in the middle of the wells. If you are sorting many plates during a day, it is advisable to calibrate a second time in between plates.
Note: If using a BD FACS ARIA, the step size in one direction might be too large to center the stream properly. In this case, you can alter the voltage on the deflector plates to adjust the location of the droplets.
- Avoid introducing contaminations while FACS sorting
This point probably goes without saying. If there is a contamination in your FACS machine, it will unfortunately also affect the sequencing data. Make sure you are using a well maintained and well-cleaned FACS machine when you are sorting into cell capture plates. Additionally, these assays are extremely sensitive to contamination, so make sure the samples are protected at all times. This means wearing gloves, a lab coat and changing these if they have been contaminated. Please reach out to our team if you need more information about cleaning procedures prior to sorting for these assays.
Plate layout
Many customers wonder in what fashion they should sort their cells; can you sort multiple cell types into one plate? Should you combine different control/experimental groups?
It is good to keep in mind that every experiment is different and that there is no standard answer to these questions. Some of our recommendations are:
- Always keep the first column of the plate empty
The wells of the first column are used for control samples and are important to assess the performance of the assay. - We usually recommend sorting one sample per plate
The batch effects of the processing are usually minimal, especially when the biology is strong. The duration of the FACS sort is more of an influence than the processing. Usually, when sorting complex plate layouts, the cell sort takes longer, which can affect RNA quality and can create batch effects between sorted plates. - Can I sort multiple cell types into one plate?
Yes, especially when dealing with primary tissue, you may simply not have another choice.