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Coating Plates with Retronectin

Created by Ravishankar Madhu

Last updated: 2023-07-10

Description

This protocol describes how to coat TC Plates with Retronectin (aka CH-296 Protein). This reagent has been shown to improve transduction efficiency of HSCs, leading it to be advantageous during gene therapy.

Reagents and Materials

Things to Note before starting

Estimated Timing

Due to the long incubation steps, the entire process takes ~3 hours to complete. Plates treated with Retronectin, can be stored at 4C for up to 1 month. In the context of a NHP Transplant, preparing these plates is best done the weekend before the Transplant occurs.

Procedure

  1. Thaw Retronectin bottle at room temperature.
  2. Sterilize the Retronectin by passing it through a syringe with a 0.22um Syringe Filter into a 50mL Centrifuge Tube.
  3. Wash the Syringe and Filter with 2 washes of 2.5mL PBS. Collect the wash into the same 50mL Centrifuge Tube.
  4. Using PBS, increase the volume of the Retronectin Solution to 50mL. This 50mL Solution of Retronectin should have a concentration of 50ug/mL
  5. Identify the surface area in cm2 of the non-TC Treated Dish you are working with.
  6. Aliquot Retronectin solution to add 10ug / cm2 to each non-TC Treated Dish. 1 2
  7. Incubate undisturbed at room temperature inside the TC-Hood for 2 hours.
  8. Aspirate off the Retronectin Solution and replace with the same volume of RM034 - List of Commonly Used Lab Buffers#2% BSA Solution|2% BSA Solution.
  9. Incubate undisturbed at room temperature inside the TC-Hood for 30 minutes.
  10. Aspirate off the RM034 - List of Commonly Used Lab Buffers#2% BSA Solution|2% BSA Solution and replace with the same volume of PBS.
  11. Wrap sealed plates with Parafilm to prevent evaporation of the PBS layer.
  12. Transfer to the cold room or a 4C Fridge until needed.

Next Steps

Coated plates can be stored at 4C for up to 1 month, but are best used within a week. Usually, Retronectin plates are used for virus transduction of Stem Cells - therefore they are used a lot in ex-vivo Transplants.

Appendix

Recipe's for Buffers

2% BSA Solution

This reagent is used for Retronectin coating plates. The solution should be kept in the TC Room Fridge.

  • 1g of Bovine Serum Albumin
  • 50mL of PBS
  • Sterilized by filtering through a 0.22um SteriFlip Filter

Footnotes

  1. As an example, a 6 well plate has a surface area of 9.6 cm2 per well. Therefore, approximately 96ug of Retronectin is recommend to coat the plate. This corresponds to approximately 2mL of 50ug/mL Retronectin Solution per well. ↩

  2. The amount of Retronectin per unit area can range from 1ug/cm2 to 20ug/cm2. I would recommend staying in the middle of the range at 10ug/cm2 ↩