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Creating CFCs Plates

Created by Ravishankar Madhu

Last updated: 2023-07-10

Description

This protocol describes how to create CFC Assay's after sorting Stem Cells. This will describe the prep work necessary before the sort, and the actual plating of the CFC plates after the sort, but will not discuss the actual Sorting for Stem Cells. For that protocol, speak with Stefan Radtke.

Reagents and Materials

  • ColonyGEL™ 1402, NHP Complete Medium
  • PenStrep
  • 5mL Syringe
  • Blunt End Needles
  • 15mL Centrifuge Tube
  • Cells that will be sorted for HSCs
  • 30mm plates
  • 60mm plates
  • 150mm plates
  • DI-H20

Things to Note before starting

Choice of Colony Gel

Different compositions of colony gel are used depending on the source of cells. 1402 is used for NHP Stem Cells, which is the main source of CFCs in the lab. If you were working with mouse CFCs speak to Heather or someone else in the Mouse Core for assistance on selecting the correct type of Colony Gel.

Estimated Timing

The ColonyGel is stored at -20C, which means the Gel has to be thawed at room temperature for ~2 hours before it can be used. Plating the collected HSCs depends on how many plates you intend to make, but on average plating 10 plates takes 30-45 minutes.

Procedure

Preparing Reagents for the Sort

Before the Sort can begin, it is important to identify how many samples will be plated. Each sample can be sorted into 4 conditions (CD34+, CD34-, CD90+, and CD45RA+). However, depending on the number of cells going into sorting, not all the samples will be sorted. In addition, conditions are usually plated in triplicate.

  1. Take the bottle of 1402 NHP ColonyGel from the -20C freezer and leave inside the TC hood over the airflow grates. Spray the sample with 70% to increase thawing speed.
  2. Once the 1402 ColonyGel is thawed, add PenStrep so that the final concentration is 1%. 1
  3. Mix the Colony Gel by rotating it in your hands. The Colony Gel is viscous, so take care.
  4. Using the autopipette (set to 3.5mL), aliquot ColonyGel + PenStrep into 15mL Centrifuge tubes.
  5. Label the aliquots with the date, Colony Gel Type, and Lot Number.
  6. Store Aliquots in the -20C for long term Storage
  7. On the day of a CFC Sort, take 1 aliquot per sort condition and thaw in the TC Hood at room temperature ~3-4 hours prior to the sort time.

After samples are sorted

  1. Calculate the total number of CFC plates that you will make. To calculate this, multiply the number of sorted aliquots by 3.
  2. Label 30mm plates from 1 to the total number you need.
  3. Create a key that details which number corresponds to which sample.
  4. Using a 5mL syringe and a blunt end needle, take up the ColonyGel, making sure to avoid any bubbles entering the syringe. The syringe should have ~3mL of ColonyGel.
  5. Add 1mL of ColonyGel into the center of 3 x 30mm plates. Avoid adding any bubbles to the plates. 2
  6. Dispose of the needle and syringe in the sharps waste container.
  7. Take the 30mm plate and rotate them in your hand to distribute the Colony Gel across the surface of the plate. Ensure that there is a continuous thin film across the entire plate. Cap the plate.
  8. Place a 60mm plate (uncapped) in the center of 150mm plate.
  9. Add 5mL of DI-H20 to the 60mm plate.
  10. Add 6 x 30mm plates with ColonyGel around the 60mm plate.
  11. Cap the 150mm plate and label the cap with the Date, Experiment ID, and your name.

Next Steps

After 10 and 14 days the CFCs need to be counted. Unfortunately i do not have the protocol for that. Speak to Greta for more information. 3

Appendix

Footnotes

  1. Usually this is 1mL per 100mL of 1402 Colony Gel ↩

  2. If you accidently add bubbles to a plate, you can suck them back up using the blunt end needle and the syringe after you finish plating all the plates. ↩

  3. Try to be avoid being the person in charge of CFC assays. It is a very time consuming and annoying. ↩