TITLE: Effective Lonza 4D Nucleofection with Inexpensive Homemade Buffers 
AUTHORS: Eric Danner

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol uses the Lonza 4D nucleofection system. Here I present protocols that reduce the price dramatically of using Lonza 4D equipment. </div><div class = "text-block">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.</div><div class = "text-block">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.</div><div class = "text-block">The homemade buffer used here was originally used and published in:</div><div class = "text-block"><a href="https://cancerres.aacrjournals.org/content/71/23/7291" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://cancerres.aacrjournals.org/content/71/23/7291</span></a><span>. It was called Amaxa V. The actual recipe used in that paper was published in the thesis here: </span><a href="https://opus4.kobv.de/opus4-fau/frontdoor/index/index/docId/2189" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://opus4.kobv.de/opus4-fau/frontdoor/index/index/docId/2189</span></a></div><div class = "text-block">Here we show a simple nucleofection of plasmid into K562.</div></div>

[STEPS]
?. [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.     ABCDEFG1Stock SolutionPreparation of Stock SolutionVol of stock to add to 15ml falcon tube2H2O6 ml3Sodium   phosphate buffer pH 7.2, 0.5 MMix 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 ml4KCl 1 M50 µl5MgCl2 1 M100 µl6HEPES 0.2 MJust dissolve in water, no pH adjustment   necessary.1 ml7Sodium   succinate 0.24 M1 ml8filter the solutionTotal volume ˜10 ml
ABCDEFG1Stock SolutionPreparation of Stock SolutionVol of stock to add to 15ml falcon tube2H2O6 ml3Sodium   phosphate buffer pH 7.2, 0.5 MMix 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 ml4KCl 1 M50 µl5MgCl2 1 M100 µl6HEPES 0.2 MJust dissolve in water, no pH adjustment   necessary.1 ml7Sodium   succinate 0.24 M1 ml8filter the solutionTotal volume ˜10 ml
?. [Prepare Reagents]
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.
?. [Prepare Reagents]
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.
?. [Prepare Reagents]
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).
?. [Prepare Reagents]
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-120This should be done quickly to remove the cells from the electroporation medium.
?. [Prepare Reagents]
Move Cells into WellAs 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)
?. [Prepare Reagents]
Wash 16-Well Strip for Re-UseTake 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.