TITLE: Murine CD8 T cell transduction
AUTHORS: Kristin Anderson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">CD8 T cells can be transduced to express genes transferred by retroviral transduction. This protocol outlines the steps for generating retrovirus from PLAT-E packaging cells to transduce primary murine CD8 T cells isolated from donor mouse spleens.</div><div class = "text-block"><span>In brief: The ectropic retroviral packaging cell line, Platinum (Plat)-E cells (Cell Biolabs, Inc.), were plated (2.2x10</span><span style = "vertical-align:super;">6</span><span> cells) in PLAT-E media on 10 mm culture plates. After 24 hours, Plat-E cells were transfected with DNA encoding the desired TCR and/or construct using the Effectene transfection kit (Qiagen). At 48 hours, media was replaced with T cell media. On days 2 and 3 post-transfection (at 72 and 96 hours) virus-containing supernatant was collected. Splenic T cells isolated from P14 Thy1.1+ female mice were stimulated with a-CD3e (clone: 145-2C11) and a-CD28 (clone: 37.51 (both from BD Pharmingen) and IL-2 (50 IU/ml Aldesleukin, UW Pharmacy), and transduced with retroviral supernatant by spinfection in polybrene (5mg/mL, 90 minutes at 1000</span><span style = "font-style:italic;">xg</span><span>) at 24 and 48 hours after T cell activation.</span></div></div>

[STEPS]
?. [Day 0: Prepare PLAT-E cells for transfection]
1. Gently remove PLAT-E media and rinse PLAT-E cells with 1x DPBS.2. Gently remove PBS and add 0.05% Trypsin. Trypsinize at 37C for 1-2 minutes or until cells being to lift off the plate with a gentle tap.3. Dilute and inactivate trypsin with 10ml PLAT-E media.Transfer cells to a conical tube, spin down @ 369xg for 4-5 minutes at 4C and remove supernatant.4. Resuspend PLAT-E cells in 10mL PLAT-E media. Count cells. Make new 10 cm plates with 2.2e6 cells per plate in 10ml PLAT-E media.Cells:Quadrants:Dilution:Calculation of concentration:Calculation of cells to add:Volume of cells per plate:Media per plate:5. Return plates to 37C incubator.DNA to transduce:Number of plates prepared:
?. [Day 1: Transfect PLAT-E cells]
1. Thaw tubes of DNA. And prepare 5mL polypropylene tubes (1/plate).2. Add 300 uL EC Buffer.3. Add 2ug DNA to EC Buffer.Record DNA amounts to add for each construct:4. Add 16uL of Enhancer and vortex for 1 second.5. Incubate 5 min at Room Temp.6. Add 60uL of Effectene. Vortex for 10 seconds.7. Incubate 10 min at Room Temp.8. Aspirate media from PLAT-E plates. Add 7mL of T cell media to each plate.9. Add 3 mL of T cell media to each tube of Effectene mixture. Pipette up and down to mix, then add slowly (dropwise) to each PLAT-E plate.10. Swirl gently to mix and return to 37C incubator overnight.
?. [Day 2: Change PLAT-E media and prepare T cells for transduction]
1. Aspirate media from PLAT-E plates and replace with 10.5 mL T cell media.2. Place the plates in the 32C incubator overnight.3. Harvest T cells by smashing donor spleens through a 40um filter. Rinse and transfer to a 15 or 50 mL tube.         Number of spleens:         Donor mice information:4. Spin down T cells down @369xg for 4-5 minutes at 4C.5. Aspirate media and lyse spleens with 2ml/spleen ACK Lysis buffer for 2 minutes.6. Dilute out the ACK Buffer with equal or greater volume of T cell media. Spin down T cells down @ 369xg for 4-5 minutes at 4C.7. Resuspend pellet in T cell media.         Volume of T cells:8. Add 1ug/ml of anti-CD3 and anti-CD28 (e.g. 20ul of Ab per 10 mL of splenocytes).         Anti-CD3 added:         Anti-CD28 added:9. Add 50 IU/mL of IL-2 (e.g. 10ul of Ab per 10 mL splenocytes).         IL-2 added:10. Transfer 10mL of splenocytes per T25 flask and place in 37C incubator overnight.
?. [Day 3: Transduction 1]
1. Transfer T cells into 50mL tubes (need 1 tube for each independent viral transduction. Number of tubes:2. Harvest virus from PLAT-E plates and filter through 0.45um Nalgene Syringe Filter.3. Add 10.5 mL T cell media per PLAT-E plate and return to 32C incubator overnight.4. Resuspend T cells in filtered viral supernatant. Calculate volume of each tube.         Volumes:5. Add IL-2 at 50 IU/mL and Polybrene at 5ug/mL.         Volume of IL-2 added to each tube:         Volume of Polybrene added to each tube:6. Plate T cell/virus mixture in 12 well plates (Add approximately 1.5-2 mL per well). Balance the plates by weight. Spin @ 1000xg for 90 minutes at 30C.7. Place plates in 37C incubator overnight.
?. [Day 4: Transduction 2]
1. Collect T cells from wells, combine similar wells into 50 mL tube, and spin down @1350 RPM for 4-5 min at 4C.2. Harvest virus from PLAT-E plates and filter through 0.45um Nalgene Syringe Filter.3. Resuspend T cells in filtered viral supernatant. Calculate volume of each tube.         Volumes: 4. Add IL-2 at 50 IU/mL and Polybrene at 5ug/mL.         Volume of IL-2 added to each tube:         Volume of Polybrene added to each tube:5. Plate T cell/virus mixture in 12 well plates (~1.5-2.5 mL per well). Balance the plates by weight. Spin @ 1000xg for 90 minutes at 30C.6. Place plates in 37C incubator overnight.
?. [Day 6: Add media (as needed) and IL-2]
1. If the wells are starting to change from oragne to yellow in color, add 1-2 mL of T cell media per well of cells.2. Add 50 IU/ml IL-2 per well.
?. [Day 7 or 8: Screen T cells for transduction efficiency]
1. Remove a small sample of cells for screening.2. Continue to add media (ass needed) and IL-2 (50 IU/ml) every 48 hours.