Hello
I am trying to electroporate DNA into freash PBMC using ECM830 Electro Square Porator (Harvard Apparatus BTX). I am running optimization with various options like voltage, pulse length and temperature. In details, my options are the below ;Cell number: fresh resting PBMC 10 million
Temperature : on ice
Cuvette : 2mm (blue cap : Lonza)
Voltage/Pulse length : 250V/20ms, 250V/10ms, 300V/10ms, 300V/5ms, 350V/5ms, 350V/2ms, 400V/2ms, 400V/1ms, 500V/1ms, 500V/0.5ms
My transfection efficiency was horrible.
I can find some papers in this field and they mostly use the program U-14 in nucleofector-I.
If possible, would you share your expertise with me ?
Thank you
Hisa
Hi,
If the efficiency of transfection PBMC is zero, consider changing the promoter in constructs.
Try to use control cell line HEK293 (which is the most often use cell line as a transfection efficiency control) or fibroblasts.
If the PBMC viability after electroporation is low, try to add to your cell culture medium ROCK inhibitor.
Good luck!
Hi Dr. Justyna,
Thank you so much.
I am using pSB-bi sleeping beauty plasmid for this purpose. (https://www.addgene.org/60511/)
Today I got about 15% transfection efficiency 24hours after electroporation.
The efficacy seems horrible, but I can expand the electroporated cells specifically.
It's OK.
But, viability is low.
Would you give me some guidance about rock inhibitor more specific ？
Thank you
Hisa
Hi,
Hi,
You can try to use ROCK inhibitor or RevitaCell™ Supplement (100X) to enhance cell viability. You should add the ROCK inhibitor or RevitaCell™ Supplement (100X) to the cells immediately after the electroporation up to 24h after transfection (then you should changes the medium on fresh without ROCK inhibitor or RevitaCell™ Supplement (100X)).
1. ROCK inhibitor, Y-27632,
https://www.stemcell.com/y-27632.html
2. RevitaCell™ Supplement (100X)
https://www.thermofisher.com/order/catalog/product/A2644501
In Annex, I attach the publication and PBMC electroporation protocol dedicated to the Neon Transfection System.
Mellott AJ, Godsey ME, Shinogle HE, Moore DS, Forrest ML, Detamore MS., Improving viability and transfection efficiency with human umbilical cord wharton's jelly cells through use of a ROCK inhibitor., Cell Reprogram. 2014 Apr;16(2):91-7. doi: 10.1089/cell.2013.0069.
https://www.thermofisher.com/pl/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system.html
Good luck!
Article Improving Viability and Transfection Efficiency with Human U...
Hi Fei
Sorry for my delayed response.
I tried RevitaCell to improve viability after electroporation, but I could not see any difference in my method.
However, I found out the optimal condition for my experience.
Among several conditions, T023 program of Amaxa works well to grow the incorporated T cells with DNA plasmid.
Thank you
Hisa
Hi Hisataka Ogawa，
These days I have tried three times to electroporate mRNA into non-activated cryo-PBMC with BTX ECM systerm, but there is nothing efficiency tested by Flow cytometry, kindly have your problem been solved? could you give me some guidence about non-activated PBMC electrporate? many thanks~@Hisataka Ogawa
Seems to work Article Development of an RNA-based kit for easy generation of TCR-e...
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