Lentiviral infection of HSCs for transplantation

*All steps involving lentivirus require double gloves and lab coat. Inactivate virus on tips, tubes, etc. with 10% bleach before discarding

  1. Coat 96 well plate with Retronectin following manufacturer’s directions.
  2. Load lentivirus onto plate following spin infection protocol in link above. Briefly, add desired volume of lentiviral sup (up to 200uL) to each well and spin at 1000-2000 xG for 2 hours at 32C to bind the virus to the retronectin.
  3. Remove the sup from the wells and replace with 100uL media. We use Stemspan serum free media supplemented with TPO(10ug/mL), SCF(10ug/mL), and pen/strep (1:100). For my stocks of TPO and SCF, use 1uL per mL of stemspan media.
  4. Sort HSCs into wells, then spin for 10 minutes, 1500 rpm at 32C to pull the cells down onto the virus. We’ve had success sorting 2000 48- LSKs per well/recipient.
  5. Incubate overnight. We try to sort in the late afternoon so that this step isn’t too long.
  6. Resuspend cells with a pipet, draw up in a syringe, then rinse the well with 30uL PBS and draw up. Keep syringes on ice, transplant immediately.