Plating 293 cells in T-25 flasks

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Plating 293 cells in T-25 flasks3 v- j" h( N' r2 ?. c
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" U, T5 A) A7 fAbout 293 cells: 293 cells are derived from human embryonic kidney cells (a.k.a., HEK 293 cells) that are stably expressing human adenovirus E1A and E1B genes. As you know, most recombinant adenoviruses are missing the E1A and E1B genes. 293 cells provide the E1A and E1B proteins and allow recombinant adenoviruses to replicate and produce more virus particles. Thus, 293 cells are also referred as the packaging line for recombinant adenoviruses. Although there are several other packaging lines available, 293 cells are the most commonly used line for generating and amplifying adenoviruses. In addition, 293 cells are also a popular choice for many transient gene expression studies because of its high transfection efficiency.
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' _2 Z/ q# Q6 J) P! q7 k& iPlate 293 cells in T-25 flasks 12-16hrs prior to transfection (approx. 40-60% confluence at transfection). Alternatively, seed 293 cells in T-25 at subconfluency (~60%) 2 ~ 4 hours prior to transfection (i.e., plating cells in early morning if you want to finish transfection within the same day). This approach has been working very well in our lab, and is particularly useful for those who are concerned about potential over-confluency of the overnight plating culture. ' N# G3 `! t4 Y/ A+ ?% C
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' Y! E- g* m1 \! k1 E' l* {7 cNote: A lower confluence could achieve higher transfection efficiency but may yield a lower absolute number of transfected cells. The following procedure can be scaled up or down proportional to the surface area of plate or well with little or no change in the results.% o! l5 L. X' e' p3 v

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II. Pac I digestion of adenoviral recombinant plasmids (i.e., pAdYFG)) B/ K, W) R- o2 r6 k

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, `; N6 c1 J0 \! a- z) n" B' fRationale: The purpose of Pac I digestion is to liberate the two ITRs of adenoviral genome so that viral DNA replication (i.e., adenovirus production) can be initiated effectively by the adenoviral Terminal Binding Protein.
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) \+ D& R9 i5 F# m) M8 uPac I digestion: you will need to perform 100ul digestion reaction, using 3ul Pac I (NE. You will need to use ~3ug DNA for the digestion. We usually use Wizard prep DNA. In this case, you may need to use approx. 30ul of DNA for Pac I digestion (as the concentration of a typical Wizard prep is ~100ng/ul). The digestion reaction should only last 30-60 min (>1 hr or overnight digestion is ridiculous and will hurt you!). [Optional: You can check 5-10ul of your digestion reaction mix (NOT your precipitated DNA) on gel to make sure that the Pac I digestion is complete]. Perform ethanol precipitation, and wash the pellet twice with 70% ethanol. Resuspend DNA in 30ul sterile ddH2O. Ready for transfection (see Stage III).
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9 u, e6 d+ y  r% kNote: 1) You should always check 1-2ul of your Wizard prep DNA on an agarose gel (and read A260, which is less reliable and optional). 2) If the concentration of your Wizard prep DNA is low, and you want to use >30ul Wizard prep for Pac I digestion, you should first precipitate DNA because the Wizard prep is usually dirty, and the impurities could inhibit your Pac I digestion. 3) There is no need to perform PC-8 (phenol/chloroform) extraction and/or gel purification of the Pac I digested pAdYFG DNA (If you do, you will get more hurt than helped)./ o. S: W" W: \5 H+ A" [/ |# E

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- R5 b5 _6 b7 K7 n4 c$ H1 HIII. Transfection of 293 cells with LipofectAMINE
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1. Prepare 1.5 ml microfuge tubes (or use the wells of a 48-well plate), the 30ul of Pac I digested DNA solution, and 200ul of OptiMEM or plain DMEM (i.e., DMEM without FBS) per transfection.
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2. Mix 200ul of OptiMEM or plain DMEN, 30ul of Pac I digested DNA, and 15ul of LipofectAMINE (Invitrogen, usually 5ul/ug DNA) per transfection. Let the mix sit in the cell culture hood for 10 min.
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" b3 v7 V8 w, K: P6 v3. Meanwhile, remove the complete medium (containing FBS) from T-25 flask, and wash cells gently by adding 5 ml of OptiMEM or plain DMEM (Note: serum free media) to the side of the flask, rocking slowly to allow the medium to cover the cells, and aspirating the medium.- h$ N8 {  U# ?  P% w- e4 y
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4. [Optional: repeat the wash once. It is NOT desirable to repeat the wash on less adherent cells, such as 293 cells].1 e# g% ~+ y. H! w8 C
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5. Add 2.5 ml OptiMEM or plain DMEM to the flask. Return flask to 37°C incubator until DNA/LipofectAMINE mixture is ready.
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6. Add the DNA/ LipofectAMINE mix to the other side of the flask. Rock gently and return flasks to the incubator.
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5 V# ^6 t( D" c7. After 2-6 hours, remove the OptiMEM or plain DMEM and replace with 8-10 ml of complete DMEM medium. [Note: longer transfection time could result in higher transfection efficiency, as long as the cells are healthy].
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8. Check GFP at 24 hrs after transfection (if you use pAdTrack, pAdTrack-CMV, or pAdTrack-TO4 based shuttle vector). Check GFP again in one week. Collect the transfected cells at 10-14 days after transfection.
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/ N+ @# o4 d# c& dVI. General comments on transfection and adenovirus making in 293 cells:
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, H. O: R' n* ^3 a& _6 y7 ?$ ^1) If a significant portion of the transfected cells is floating at the end of your transfection, do not remove Lipo/DNA-containg medium. Instead, just add 6.0ml of complete DMEM medium, and replace it with fresh complete DMEM medium next morning.7 O* i7 j1 S2 l2 @; S) t* E
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2) The transfected cells usually become confluent at 2-3 days after transfection, and will stay confluent until you collect them. The culture medium usually becomes yellowish. Do not panic! There is No Need to change the medium [If you do, you will get hurt as the generated adenoviruses are usually released to the medium, and subsequently infect more 293 cells, leading to higher virus titers]. Instead, you can add 1-2ml of the complete DMEM medium every 3-5 days. We usually do not add any fresh medium or only add 2ml fresh complete DMEM at day 10.
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: ~. m9 h/ ^* M3) You should see some intensified GFP foci usually at 5-7 days after transfection. If your transfection efficiency is not very high (i.e., <10%), you may want to leave the transfected cells for up to 20 days in order to obtain higher titers of the initial virus lysate. Waiting for 1-2 more days makes a big difference in terms of enhancing initial virus titers. Patience will pay off as it takes much longer time to amplify low titer viral lysate!

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