1 2 To begin, see the HEK 293T cells3 24 hours before transfection 4 in a 15 centimeter dish containing GMEN. 5 Incubate the cells at 37 degrees Celsius 6 and 5%carbon dioxide 7 until they reach 40 to 50%confluency. 8 Label three 15 milliliter plastic tubes 9 with the appropriate lentivirus name.
10 Add 1.710 milliliters of reduced serum medium 11 and 90 microliters of transfection reagent to each tube. 12 Mix the contents by vortexing 13 before incubating the tubes at room temperature. 14 After five minutes, add the packaging vector mixture 15 to the transfection medium and vortex.
16 Then add 7.5 micrograms of reprogramming vector 17 and the pWPT-GFP control 18 to the respective transfection mixture. 19 Vortex the tubes for two seconds 20 before incubating for 15 minutes at room temperature. 21 To transfect the 293T cells with each lentivirus, 22 dropwise, add a transfection DNA mixture into the dish.
23 Incubate the transfected cells at 37 degrees Celsius 24 and 5%carbon dioxide. 25 After 14 to 16 hours, 26 replace the medium with 30 milliliters 27 of fresh 293T medium 28 and continue incubation for 60 to 72 hours. 29 Observe the cells daily 30 and check transfection efficiency.
31 Next, transfer the virus transfection culture 32 into 15 milliliter tubes 33 and spin down at 1, 932 G for 10 minutes 34 at four degrees Celsius. 35 Filter the lentiviral supernatant 36 using a 0.45 micron syringe filter 37 into a new 50 milliliter tube. 38 One day before lentivirus transduction, 39 prepare two wells of a 6-well plate 40 containing 100, 000 PDAC cells per well.
41 Prepare two 15 milliliter tubes 42 with two milliliters of pre-warmed PDAC medium. 43 In the first tube, 44 add 12 milliliters of reprogramming lentivirus. 45 Then add 12 microliters of polybrene 46 and mix by pipetting.
47 To the second tube, 48 add two microliters of polybrene. 49 Now carefully remove the medium from each well 50 of a 6-well plate 51 and wash once with PBS at room temperature. 52 Add the reprogramming infection medium 53 from tube one to the first well.
54 Add the mixture from tube two to the second well. 55 Incubate the cells overnight at 37 degrees Celsius 56 with 5%carbon dioxide and 5%oxygen. 57 The next day, replace the medium 58 from both wells with fresh PDAC medium 59 and continue incubation 60 for up to 48 hours as demonstrated.
