1. Vector: 30 ug purified pBabe-puro and target plasmid(s) each in TB.
2. 2X HeBS

3. 2.0 M CaCl₂
4. Chloroquine stock
5. Bosc23 Cells
6. DMEM, with 10% Fetal bovine serum and 1% penicillin-streptomycin stock
7. Trypsin-EDTA
8. Sterile MilliQ H2O
9. Pipettes and tips (20, 200 and 1000 ul)
10. 10 ml sterile syringes (luer lock-type prefered)
11. 0.45 (or 0.22) micron, low protein-binding, surfactant-free syringe filters
12. Freezer vials

Day 1, AM-PM: 

1. Plate 7.5X10⁶ Bosc23 cells into 12 mls of 10% FBS DMEM (for 10cm dish) and plate at 37^oC incubator for 24 hrs prior to transfection.

2. 1 hr prior to transfection, remove media and add 12 mls of fresh media.
3. 15 min prior to transfection, add 12 ul of chloroquine (Sigma C6628, 25mM in PBS, 1000X).
4. Prepare a transfection cocktail by adding
   • 30ug retroviral vector plasmid DNA to water and dilute to 1314ul
   • 186ul of 2M CaCl2 (Sigma C7902) to the DNA solution
   • 1.5ml 0f 2X HeBS
   Immediately mix and add this solution into the cells, then gently swirl the plate to ensure uniform mixing.

Day 2, night: 

5. Remove media (with chloroquine) ~ 10 hrs after transfection and gently replace 12 mls of fresh media.

Day 3, PM: 

6. 36 hrs after transfection, refeed the cells with 9 mls of media (the transfected cells should be close to 100% confluent at this time). If the cells are less than that, let them sit for another 12 hrs.

Day 6, AM:

7. Collect retroviral supernatant (60 hrs to 72 hrs following transfection) by filtering the supernatant through a 0.45um filter.
8. Either aliquote and freeze supernatants in appropriate volumes for subsequent infection.