Lipofectamine 3000 Protocol Written Out With Timing Specifics Regarding the Tran - (Aug/13/2016 )
I am a high school student working in a very new lab consisting of my PI and myself. I am struggling to meet a close deadline for my project and cannot afford human error at this stage. Could someone please help with some reccomendations? I will also be posting in the ELISA assay forum so if you could, find me there as well.
Thank you all for your help,
Maximilian Bazil
https://tools.thermofisher.com/content/sfs/manuals/lipofectamine3000_protocol.pdf
Day 0
1) "Seed cells to be 70–90% confluent* at transfection"
You will know how fast your cells grow in your medium when passaged into a T-Flask from prior experience. If you do not have this prior experience you will need to look at published literature. You can also calculate the surface area of a 96-well, 24-well, or 6-well plate (area of a circle equation) versus the surface area of the T-flask you are using. See here for precalculated surface areas and number of cells at 100% confluency for HeLa cells: https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/cell-culture-useful-numbers.html
So let's say your cells double once every 24 hours in the T-flask immediately after splitting*. The T-flask has a surface area of X, the well of the transfection plate has a surface area of 1/8th X. From a 70%-90% T-flask you would plate 1/16th of the total trypsinized volume into the well (assuming zero death from the trypsinizing and scraping, which isn't going to happen). Or ideally you'd place a known quantity of cells from a suspension culture since many fewer of them will be dead than after passage from a T-flask. You'd want to place enough cells to make your transfection well ~35%-45% confluent since you know that they will double to ~70%-90% confluent in 24 hours. You let them adhere to the surface for an hour or so and then swap the media with fresh media so that they will grow well (tip the well to the side and carefully pipette off the medium from the side of the well so that you don't disrupt the cells. Add fresh medium very soon after pulling the old medium off).
If you do not know how fast your cell line doubles on a surface, to make everything simple I'd plate multiple wells of your cell line in a dilution series (e.g. 2x10^5 cells, 1.8x10^5, 1.6x10^5, 1.4x10^5, 1.2x10^5, 1.0x10^5, 0.8x10^5, 0.6x10^5, 0.4x10^5, 0.2x10^5 cells per well) in order to ensure that you will have at least one well around 70%-90% confluence on Day 1. I'd do this dilution series in duplicate or triplicate, and would likewise do the transfection in duplicate or triplicate, just to be safe.
* - "Confluence" is how much of the surface of the well is covered with cells. See here: http://www.essenbioscience.com/media/uploads/files/HT1080_Timecourse_Plus_Images.jpg where the 6h time point is ~25% confluent, the 24h is ~50% confluent, and the 48h is >95% confluent.
* - "Splitting" is when you trypsinize and scrape a T-Flask and then place a sub-portion of this into a new flask or suspension culture, or when you cell count from a suspension culture and then place a portion of that suspension culture into a T-flask or new suspension culture.
Day 1 - This entire process should take 30 minutes to an hour, and will be done about 24 hours after the cells were seeded.
!!!!Make sure the DNA you are transfecting is relatively free of endotoxins (by using an "endotoxin-free" DNA prep kit, or an endotoxin removal kit), and is free of other contaminants. As a precautionary measure it is a good idea to pass it through a sterile 0.22 um syringe filter into a new, sterile microcentrifuge tube, while in the laminar flow hood. The DNA concentration will stay mostly the same (a little loss), but you will lose some volume. I'd have at least 100 microliters of DNA and expect 50 - 60 microliter after syringe filtration. Make sure to "blow out" the syringe filter (unscrew the filter, pull out the plunger, rescrew the syringe filter, and plunge once more) to get as much of the DNA out as possible. Be careful that you do not push the DNA out fast enough that it ricochets out of the new tube!!!!
"Dilute Lipofectamine™3000 Reagent in Opti-MEM™ Medium (2 tubes), mix well"
Follow the instructions as indicated. You can generally use other serum-free* medium instead of Opti-MEM. The instructions are recommending that you do this in duplicate with the two indicated different concentrations of Lipofectamine 3000.
"Prepare master mix of DNA by diluting DNA in Opti-MEM™ Medium, then add P3000™ Reagent, mix well"
Follow the instructions.
"Add Diluted DNA to each tube of Diluted Lipofectamine™ 3000 Reagent (1:1 ratio)"
Follow the instructions.
"Incubate - Incubate for 10–15 minutes at room temperature"
Follow the instructions.
"Add DNA-lipid complex to cells"
Follow the instructions.
* - "Serum-free" means it does not contain Fetal Bovine serum.
Day 2-4
"Visualize/analyze transfected cells - Incubate cells for 2–4 days at 37°C. Then, analyze transfected cells."
Follow the instructions. You'll wait 1 to 3 days after transfection before analyzing the cells. Analyze the cells whichever way you need to for whatever it is you transfected them with. If you can see successful transfection through a visual diagnostic (e.g. the cells change color) do this every days. If you cannot see this, but need to run SDS-PAGE + Coomassie stain or Western blot, I'd have multiple replicates of each transfection and harvest a replicate on each of these three days. You can resuspend* in gel loading dyesample buffer, boil, and then freeze this so that all samples can be loaded on the same gel on Day 4 or 5.
* - To resuspend for SDS-PAGE: Remove all media from the well (slightly angle the well so the medium is on an edge. Use a pipette tip to pipette off the media without disrupting the cells. Add sample buffer and scrape the cell with the pipette tip while mixing them with the sample buffer until all have been resuspended, then pipette this into a microcentrifuge tube.
I have no input about the ELISA question. I'd recommend contacting the manufacturer.