Welcome guest, Sign in

Home

X
Loading

293T and Pheonix cells grow in DMEM + 10%FBS. If you are transfecting other cells, you can use whatever medium those cells normally grow in and change to DMEM + 10%FBS on the day of the transfection. You can change back to “normal” medium 24 hours post-transfection. Calcium Phosphate transfection of M2182 in RPMI (w/o FBS) was reported to cause cells to die. All the transfection have been done in DMEM + 10% FBS, no transfection in other media was performed so far.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

[Bio101] Lentivirus and Retrovirus Transfection

Molecular Biology > DNA > Transfection
Author: Yanlin Huang
3/5/2011, 20886 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.38

[Abstract] 293T and Pheonix cells grow in DMEM + 10%FBS. If you are transfecting other cells, you can use whatever medium those cells normally grow in and change to DMEM + 10%FBS on the day of the transfection. You can change back to “normal” medium 24 hours post-transfection. Calcium Phosphate transfection of M2182 in RPMI (w/o FBS) was reported to cause cells to die. All the transfection have been done in DMEM + 10% FBS, no transfection in other media was performed so far.

Materials and Reagents

  1. 293T and Pheonix cell line
  2. 1x Dulbecco's modified eagle medium (DMEM) (high glucose, liquid) (Life Technologies, InvitrogenTM)
  3. Fetal bovine serum (FBS) (Hyclone)
  4. Na2HPO4
  5. NaCl
  6. TE
  7. HEPES (Sigma-Aldrich, catalog number: H7006 )
  8. Packaging vector PcmvR8.74
  9. Envelop vector PMD2VSVG
  10. Reporter vector DsRED, GFP or LacZ
  11. 2x HBS (see Recipes)
  12. 2 M CaCl2 (Covidien, MallinkrodtTM, catalog number: 4160) (see Recipes)

Equipment

  1. Tissue culture incubator
  2. Tissue culture plates of the appropriate size (Thermo Fisher Scientific)
  3. 6 cm or 6 well plates
  4. Microscope
  5. 0.45 μm filter
  6. p1000 pipettor

Procedure

  1. Day 0
    Plate 4.5 x 106 293T (or Pheonix for retrovirus) on 10 cm plate. Incubate overnight at 37 °C . You can also scale down to 6 cm or 6 well plates.
    Note: Alternatively, you can plate the cells the same day as the transfection. See Day 1.

  2. Day 1: Transfection
    Note: If you plated cells on the previous day, change medium to DMEM + 10% FBS 2 h prior to transfection and skip to step 4.
    1. Plate 293T (or Pheonix for retrovirus) at ~50% confluence on 10 cm TC plates. I calculate it roughly from the original plate confluence. Example: I will plate 2 x 10 cm plates from 100% confluence 10 cm plate. I have scaled down to 6 cm and 6 well plates. Just keep final confluence of cells at roughly 50%.
    2. Incubate plates in 37 °C incubator for ~3 to 5 h to allow cells to attach.
    3. Thaw out all reagents to room temp before proceeding with transfection.
    4. Once cells attached, prepare DNA mix for transfection. For each 10 cm plate, add the following to 5 ml polypropylene tube:
      1. 10 μg DNA of interested (for retrovirus, 10 μg DNA)
      2. 6.5 μg packaging vector (packaging vector already in Pheonix, so no need to add)
      3. 3.5 μg envelope vector (for retrovirus, 5 μg envelope vector)
      4. 0.1 to 0.2 μg of reporter vector (optional) - GFP, DsRED, or LacZ
      5. Bring mixture up to 437.5 μl with 0.1% TE in H2O
      If you use different size plates, just scale down. See table below.
      Notes: 
      1. You can make a master mix if you have multiple plates of the same transfection. However you need to do each transfection separately for high efficiency.
      2. The ratio of DNA: packaging vector: envelope vector is crucial for max viral titer. The ratio provided here was determined by the Weissman Lab. Please keep to this ratio as much as you can.
    5. Vortex the DNA mixes on highest speed setting and add 62.5 μl of 2 M CaCl2. While still vortexing, add 500 μl 2x HBS drop wise to the DNA/CaCl2 mix (roughly 2 drops/sec using p1000 pipettor).
    6. Immediately add HBS/DNA solution onto cells. Do this in a gentle, drop wise manner and spread it across cells in medium.
    7. In a few minutes you should be able to observe, under microscope, evenly distributed small black particles on top of the cells.
    8. Incubate cells in 37 °C incubator overnight.


      2x HBS
      2 M CaCl2
      DNA mix
      DNA: Pack: Env
      10 cm plates
      500 μl
      62.5 μl
      437.5 μl
      10 μg: 6.5 μg: 3.5 μg
      6 cm plates
      250 μl
      31 μl
      219 μl
      5 μg: 3.3 μg: 1.8 μg
      6 wells plate
      83 μl
      10.5 μl
      73 μl
      1.5 μg: 1 μg: 0.6 μg

  3. Day 2: 24 h post-transfection
    1. Change medium to 10 ml fresh DMEM 10% FBS. You can generally see the efficiency of your transfection by now if you used a reporter vector. At 0.2 μg of DsRED, I can usually observe >75% transfection efficiency. If you can’t see much fluorescent cells at this time, wait until 48 h post-transfection and observe again.
      Note: We have always packaged our viruses in 37 °C incubator although it has been reported that the virus is more stable if incubation is carried out at 32 °C .
    2. If you want to titer the virus, plate target cells now for infection the next day. I often plate 3 x 10 cm plates per virus for 3 different titrations.

  4. Day 3: 48 h post-transfection
    1. Aliquot supernatant of transfected cells into desired volume and freeze at -80 °C to kill any cells in the supernatant. Alternatively, you can filter the supernatant through a 0.45 μm filter to remove the cells. You can also choose to centrifuge the supernatant first if there are many floating cells. However, you need to either freeze or filter the supernatant before using the virus to completely remove any chances of cell contamination.
    2. Store viruses at -80 °C . 
      If you are titering or infecting target cells, follow the rest of the protocol.
    3. Add 10 μl 1,000x polybrene (1,000x = 5-8 μg/ml) to each 10 cm plate of target cells (10 ml medium). Add 0.1 μl, 1 μl, or 10 μl of virus supernatant to each plate of cells for titering your virus. Incubate at 37 °C overnight.

  5. Day 4: Remove virus supernatant
    1. 24 h post-infection, change to fresh medium. Incubate overnight at 37 °C .

  6. Day 5: 24-48 h post-infection
    1. Cells are now ready to be assayed for any biochemical event of interest (example: start selection with appropriate antibiotic). The actual reverse transcription and integration take place within 24-36 h, depending on cell growth kinetics.
    2. Culture cells as normal.

Recipes

  1. 2x HBS for calcium phosphate coprecipitation transfection
    1. Make stock sln of Na2HPO4 dibasic (5.25 g in 500 ml H2O)
    2. Make 2x HBS
      8.0 g NaCl
      6.5 g HEPES
      10 ml Na2HPO4 stock solution
    3. Bring volume close to 500 ml. Divide into 3 batches and pH each to 6.95, 7.00, and 7.05. Test each batch using LacZ, DsRed, or GFP to see which pH gives best transfection efficiency.
  2. 2 M CaCl2
    1. Add 14.702 g of CaCl2·2H2O to 50 ml H2O. 
    2. F.W. of CaCl2·2H2O = 147.02 g. -- > 2 M= (2 mol/L) (147.02 g/mol)
    3. CaCl2 is from Mallinkrodt (catalog number: 4160). It is important you use their CaCl2.


How to cite: Huang, Y. (2011). Lentivirus and Retrovirus Transfection. Bio-protocol Bio101: e38. DOI: 10.21769/BioProtoc.38; Full Text



Share Your Feedback:

  • Add Photo
  • Add Video

Bio-protocol's major goal is to make reproducing an experiment an easier task. If you have used this protocol, it would be great if you could share your experience by leaving some comments, uploading images or even sharing some videos. Please login to post your feedback.

Ask the Authors:

  • Add Photo
  • Add Video

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.


Login | Register
1/10/2015 8:04:48 PM  

Syed Raza
Unversity of Utah

Hi i have a question:
How to know what ratio should I took of DNA/envelope/packaging vector for my target vector of 14kb which is a second generation compatible. I tried with 12.5:12.5:5 (DNA:Packaging:Envelop) for 150mm dish, and importanly, can i take the same ratio (12.5:12.5:5) and divide it for 6 well plate as the surface area of the 150mm plate is approx 152 & of 6 well plate is 9.5. If not then how this ratio has to be decided. for e.g. how in your protocol it is adjusted to 167.5ul (total) of 1.5μg:1μg:0.6μg. please kindly explain. My Vector is of 14 kb. Thanks

Syed
Syedshadabraza@gmail.com

1/10/2015 8:05:28 PM  

Syed Raza
Unversity of Utah

my mail id is syedshadabraza@gmail.com

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register

9/23/2013 11:04:22 AM  

weicheng chang
UC berkeley

Hi:
May I ask a question about the difference between Lenti virus and Retrovirus? I want to pack retrovirus via a viral plasmid backbone "FUGW" which is designed for Lenti virus package. Do you think it will be work or not? Are there any difference sequence design for Lenti and retro virus plasmid backbone? Thanks very much.

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register

5/31/2011 9:18:24 PM  

Yuanqing Lin

If i get 83% transfection of 293T cells by Flow what is my titer?

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register

How to cite
Share
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook