Apr 17, 2020
Effective Lonza 4D Nucleofection with Inexpensive Homemade Buffers
- Eric Danner1
- 1Max Delbrück Center
Protocol Citation: Eric Danner 2020. Effective Lonza 4D Nucleofection with Inexpensive Homemade Buffers . protocols.io https://dx.doi.org/10.17504/protocols.io.64mhgu6
Manuscript citation:
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License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 04, 2019
Last Modified: April 17, 2020
Protocol Integer ID: 27501
Keywords: Nucleofection, Homemade Buffers, 4D, Lonza 4D
Abstract
This protocol uses the Lonza 4D nucleofection system. Here I present protocols that reduce the price dramatically of using Lonza 4D equipment.
1. The 16 well strip kits are expensive. These can be washed and re-used. This allows a single strip for hundreds of nucleofection experiments.
2. The nucleofection buffers are limited (P1, P2, etc). However we have used home made buffers that effectivly target many primary cell types. We have used this Cas9 RNPs (protein/RNA) and plasmid delivery. We have used this in T and B cells, cell lines, and more.
The homemade buffer used here was originally used and published in:
https://cancerres.aacrjournals.org/content/71/23/7291. It was called Amaxa V. The actual recipe used in that paper was published in the thesis here: https://opus4.kobv.de/opus4-fau/frontdoor/index/index/docId/2189
Here we show a simple nucleofection of plasmid into K562.
Guidelines
There are many options for buffers to try for the nucleofection:
1. You can use the purchased buffers from Lonza
2. Use our AmaxaV buffer published here.
3. For some cells Opti-MEM works well and should be tried.
4.The Porteus Lab also has a homemade buffer that they have published:
- Solution I: Dissolve 2g ATP•Disodium Salt and 1.2g MgCl2•6H2O in 10mL nuclease-free UltraPure water, filter sterilize, make 20µL aliquots, and store at -20°C for up to two months.
- Solution II: Dissolve 6g KH2PO4, 0.6g NaHCO3, and 0.2g glucose in 500mL nuclease-free UltraPure water, adjust pH to 7.4, filter-sterilize, make 1mL aliquots, and store at 4°C for up to 3 months.
- Mix 20µL solution I with 1mL solution II (see Reagent Setup). This makes for 10 electroporations (100µL each) b
Benifit::
The benefit of homemade buffers/ strip re-washing is it becomes inexpensive to scale up experiments. This includes initial optimization of the Lonza program chosen. The published programs are not always the best. Do trials to look at cell death and nucleation efficiency.
Materials
MATERIALS
Sodium phosphate monobasic monohydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9638
HEPESMerck MilliporeSigma (Sigma-Aldrich)Catalog #H6147
Sodium succinate, dibasic, hexahydrateBio Basic Inc.Catalog #SB0889.SIZE.500g
Magnesium chloride hexahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M2670
Gibco Penicillin-Streptomycin (10,000 U/mL) (Pen/Strep)Fisher ScientificCatalog # 15-140-122
FBSInvitrogen - Thermo Fisher
Potassium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9333
Disodium phosphateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S7907
IMDMGibco - Thermo Fisher ScientificCatalog #12440053
4D-Nucleofector System with X UnitLonzaCatalog #AAF-1002X
4D-Nucleofector® X Kit S (32 RCT) specific for cell typeLonzaCatalog #V4XC-1032
Prepare Reagents
Prepare Reagents
Make Nucleofection Buffer (AmaxaV):
Stocks can be stored RT for a long time (we use ours longer than 6 months). Making working solutoin from stock solution, vacuum filter through 0.45um filter. This should be stored at 4C. Discard after 3 months or when you see white precipitate.
| Stock Solution | Preparation of Stock Solution | Vol of stock to add to 15ml falcon tube | |||||
| H2O | 6 ml | ||||||
| Sodium phosphate buffer pH 7.2, 0.5 M | Mix 7 parts of 1 M NaH2PO4 with 18 parts of 1 M Na2HPO4 and 25 parts of water. Check pH. If pH needs to be adjusted, use the two phosphate stock solutions and not HCl or NaOH. (7ml + 18ml + 25ml = 50 ml) | 1.8 ml | |||||
| KCl 1 M | 50 µl | ||||||
| MgCl2 1 M | 100 µl | ||||||
| HEPES 0.2 M | Just dissolve in water, no pH adjustment necessary. | 1 ml | |||||
| Sodium succinate 0.24 M | 1 ml | ||||||
| filter the solution | Total volume ˜10 ml |
Prepare for Nucleofection:
This nucleofection conditions us 2 ug of plasmid (Cas9 plasmid is about 9kb). Smaller plasmid nucleofect easier. If the plasmid has a fluorescence marker or some other signal, do a titration experiment to see your ideal amount (0.2- 4ug).
Add the plasmid for each condition to a well in the pcr strip. Ideally the volume is less than 5 ul for the Londza 4D 16-well strips, but I have added up to 10 ul of plasmid solution successfully (final reaction condition would then be 20 ul of cells + 10ul plasmid solution). Plasmids should be in TE or water.
Count and pellet K562 Cells
*cells should be in log phase of growth*
Use 500k K562 cells/ reaction well. (1 million also works fine)
Take the total cells needed for all reactions. Pellet at 300 rcf for 4 min.
Re-suspend in AmaxaV:
Remove supernatant from cells.
Add 20ul AmaxaV buffer for each 500k cells and re-suspend.
i.e. for 6 reactions take 3 million cells, re-suspend in 120ul LonzaBuf1 (AmaxaV should be room temp).
Nucleofect
Add 20ul AmaxaV/Cell solution onto the side of each of the PCR tubes. After adding cells to the side of the PCR tube. Tap the PCR tube on the hood bench until cells fall into the bottom and mix with the pre pipetted plasmid.
Using multi-channel pipette mix 2-4x. Transfer the cells/DNA to the 4D 16-well strip. Tap strip so that there is no air bubbles at the bottom.
Nucleofect with FF-120
This should be done quickly to remove the cells from the electroporation medium.
Move Cells into Well
As quickly as possible after nucleofection add 70-80ul IMDM/10%FBS/1%PenStrep to each well of the strip (nucleofected cells/DNA/buffer solution). Or opti-mem if using other cell types. Then transfer the entire 100ul solution to a well in a 12-well plate containing 1ml of IMDM/FBS/PenStrep media.
Selection media can be added in 16 hr (next morning)
Wash 16-Well Strip for Re-Use
Take Lonza 16-well strip and rinse with DI water throughly. Place in 50ml falcon tube. Fill falcon and strip with 70% ethanol. Leave for 5 min. Then put strip on bench in hood to dry. Can also leave it in 70% ethanol and dry directly before using.
16 well strips can then be used 40x or more times.
We have seen no issue with cross contaminations of plasmid from one experiment to another, though in principle that could happen.