Skip to content

Staining Cells for Flow

Created by Ravishankar Madhu

Last updated: 2023-07-10

Description

This protocol outlines how to stain cells in preparation for Flow Cytometry.

Reagents and Materials

A group of antibodies used in conjunction to perform analysis is referred to as a panel. Depending on your project's scope, different panels might be called for. Some common ones are listed below: - RM007b - NHP HSC Panel - RM007a - NHP Lineage Panel

  • Compensation Beads

Things to Note before starting

Estimated Timing

After the samples have been stained, they need to be incubated for approximately 30 minutes. 1

Procedure

For Sample going to be run on an Analyzer

  1. Prepare and label 1 FACS tube for each Flow Condition and 1 FACS tube per antibody in the panel
  2. Aliquot 1e6 cells into FACS tubes. 2
  3. Add 2 drops of compensation beads into the FACS tubes labeled for each individual antibody
  4. Add the respective antibody panel in accordance with their respective dose (amount of antibody to add per 1e6 cells).
  5. Add a dose of antibody to the respective compensation FACS tube.
  6. Wrap the FACS tubes in aluminum foil and incubate at room temperature for 30 minutes. 1
  7. Wash cells with PBS (using the squeeze bottle located next to the centrifuge).
  8. Spin down at 800 x g for 5 minutes. A small pellet should be seen at the bottom of the tube. 3
  9. Decant the PBS by pouring it into the sink. Take care to not disrupt the pellet.

For Samples going to be run on the Sorter

The main difference between preparing samples for the Cell Sorter is that you tend to load more cells. Therefore, you should scale up the doses of the antibody to account for the increase in cells. Otherwise, the protocol is identical.

Next Steps

Take the samples down to the flow core and run them on the appropriate machine. Speak with Stefan to get specific instructions on how to operate the flow machinery.

Appendix

Footnotes

  1. While the recommending incubation is 30minutes, you can get away with staining the samples for as little as 10-15 minutes if you are short of time. ↩↩

  2. The volume for the aliquot should not be smaller than 20uL. So if your stock cell concentration is too high, diluting the sample before making your aliquot will be useful. The aliquot should also not contain more than 200uL of volume as that can interfere with antibody binding. The ideal volume should be somewhere between 50uL and 150uL. ↩

  3. If you do not load a lot of cells then the pellet might not be a discrete white pellet. It might look like a hazy cloud around the bottom of the tube. ↩