Jan 23, 2017
Electroporation of COS-7 cells and functionalization of QDs
- Eugene A Katrukha1,
- Marina Mikhaylova1,
- Hugo X van Brakel1,
- Paul M van Bergen en Henegouwen1,
- Anna Akhmanova1,
- Casper C Hoogenraad1,
- Lukas C Kapitein1
- 1Utrecht University
External link: http://biorxiv.org/content/early/2016/11/23/089284
Protocol Citation: Eugene A Katrukha, Marina Mikhaylova, Hugo X van Brakel, Paul M van Bergen en Henegouwen, Anna Akhmanova, Casper C Hoogenraad, Lukas C Kapitein 2017. Electroporation of COS-7 cells and functionalization of QDs. protocols.io https://dx.doi.org/10.17504/protocols.io.g2mbyc6
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
Created: January 23, 2017
Last Modified: March 23, 2018
Protocol Integer ID: 4909
Abstract
Detailed cell electroporation protocol from paper:
Probing cytoskeletal modulation of passive and active intracellular dynamics using nanobody-functionalized quantum dots
Eugene A Katrukha, Marina Mikhaylova, Hugo X van Brakel, Paul M van Bergen en Henegouwen, Anna Akhmanova, Casper C Hoogenraad, Lukas C Kapitein
Guidelines
From our experience, the number of incorporated QDs per cell mainly is proportional to the pouring pulse voltage, length (duration) and repetition. At the same time increase in these values leads to the overall decreased cell survival.
Another improvement can come from increasing QD concentration in the final electroporation solution. For example, by reducing Ringer's volume. But in this case air discharge can happen if electrodes are not immersed in solution.
18mm coverslips and 12 well cell culture plates can be also used.
General advices if using different electroporator:
Specific CUY900-13-3-5 cell-culture-plate electrode has 5 mm distance between electrodes. What is important, is voltage per cm value. In our case, for pouring voltage V=200 V, l=0.5 cm, i.e. V/l = 400 V/cm.
Materials
STEP MATERIALS
PBS - Phosphate-Buffered Saline (10X) pH 7.4Thermo Fisher ScientificCatalog #AM9625
Qdot 625 Streptavidin ConjugateThermo Fisher ScientificCatalog #A10196
PBS - Phosphate-Buffered Saline (10X) pH 7.4Thermo Fisher ScientificCatalog #AM9625
Qdot 625 Streptavidin ConjugateThermo Fisher ScientificCatalog #A10196
Protocol materials
PBS - Phosphate-Buffered Saline (10X) pH 7.4Thermo Fisher ScientificCatalog #AM9625
Qdot 625 Streptavidin ConjugateThermo Fisher ScientificCatalog #A10196
PBS - Phosphate-Buffered Saline (10X) pH 7.4Thermo Fisher ScientificCatalog #AM9625
Qdot 625 Streptavidin ConjugateThermo Fisher ScientificCatalog #A10196
PBS - Phosphate-Buffered Saline (10X) pH 7.4Thermo Fisher ScientificCatalog #AM9625
Qdot 625 Streptavidin ConjugateThermo Fisher ScientificCatalog #A10196
Plate cells
Plate cells
Plate COS-7 cells using 24 or 25 mm coverslips and 6-well Corning Costar plates (Sigma #CLS3516) .
Perform this step one day in advance for non-functionalized QD electroporation.
If using GFP-proteins transfection, plate them 2 days in advance and 1 day in advance transfect them to allow GFP-protein expression.
Prepare QD - GFP-nanobody mix
Prepare QD - GFP-nanobody mix
One day in advance prepare mix of QD with biotinylated GFP-nanobody (bio-VHH_GFP):
Per each cell coverslip/well prepare one vial with:
- 2 µL of Qdot 625 streptavidin conjugate (Thermo Fisher #A10196, stock concentration 1 µM)
- 20 µL of bio-VHH_GFP (purified, concentration 0.7-0.8 µg/µl)
- 187 µL of PBS
In case of electroporation without GFP transfection, prepare 2 µL of Qdot 625 with 198µL PBS mix immediately before step 5.
Instead of biotinylated GFP-nanobody purification, one can perform biotinylation of commercially available GFP-nanobody (Chromotek gt-250) http://www.chromotek.com/products/nano-traps/gfp-trap/gfp-binding-protein/
PBS - Phosphate-Buffered Saline (10X) pH 7.4Thermo Fisher ScientificCatalog #AM9625
Qdot 625 Streptavidin ConjugateThermo Fisher ScientificCatalog #A10196
Incubate QD - GFP-nanobody mix at RT
Incubate QD - GFP-nanobody mix at RT
Incubate vials for 1 hour at room temperature.
01:00:00
Incubate QD - GFP-nanobody mix at 4C
Incubate QD - GFP-nanobody mix at 4C
Incubate vials at 4°C overnight (~12 hours)
12:00:00
Preparation of electroporation (plate
Preparation of electroporation (plate
Pre-heat Ringer's solution (10 mM Hepes, 155 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 2 mM NaH2PO4, 10 mM glucose, pH 7.2) at 37C water bath.
In the absence of Ringer's, cell culture medium or PBS can be used, but with lower electroporation efficiency.
In one well of new 6-well Corning plate add 1.8 ml of warm Ringer’s solution and 200 µl of QD - GFP-nanobody mix.
It is convenient to put 70% ethanol and water in the neighbor well to wash electrode.
Preparation of electroporation (Electroporator)
Preparation of electroporation (Electroporator)
We used NEPA 21 electroporator http://www.nepagene.jp/e_products_nepagene_0001.html
with CUY900-13-3-5 cell-culture-plate electrode http://www.nepagene.jp/e_products_nepagene_0008.html
Setup electric pulse parameters:
For poring pulse:
Voltage (V) 200
Length (ms) 5
Interval (ms) 50
Number 2
Decay Rate (%) 10
For transfer pulse:
Voltage (V) 50
Length (ms) 50
Interval (ms) 10
Number 10
Decay Rate (%) 40
Make sure your electrode is not covered with rust, clean it.
Preparation of electroporation (Cells)
Preparation of electroporation (Cells)
With tweezers remove a coverslip with cells and put it into plate
with well with warm Ringer's and QD-nanobody mix.
Preparation of electroporation (impedance check)
Preparation of electroporation (impedance check)
Insert electrode into well with cells coverslip, immersing it in Ringer's solution, so it touches the coverslip.
Press Ω button to measure impedance.
Measured impedance will be displayed in the Impedance indow (in kΩ).
Make sure that it is less then 1 kΩ (0.1-0.2 is fine).
If values of impedance around 30-60 kΩ, add more RInger's solution.
This step is to make sure that all surfaces of electrode are fully immersed into water and electroporator will not discharge into air.
Electroporation (1)
Electroporation (1)
Once impedance is below threshold, press the "Start" button (or pedal, if it is equipped)
and hold electrode still for the program to finish (1-2 seconds).
During charging and pulsing, a warning beep will sound. When program is complete, a short double beep will sound.
Electroporation (2)
Electroporation (2)
Rotate electrode 90 degrees around its axis, measure impedance (step 8) and run electroporation program again ('Start' button/pedal).
This step sends charge in the perpendicular direction, increasing electroporation efficiency.
Cells wash
Cells wash
Using tweezers, move cells to an empty well of 6-well plate and wash it 3-4 times with warm PBS or cell culture medium to remove QD in solution.
Electrode wash
Electrode wash
Wash electrode in neighbor wells (or any other volume) in water, ethanol and another round of water. Dry it with a napkin and wrap in plastic to prevent rusting.
Cell mounting for imaging
Cell mounting for imaging
Mount cells in any live-imaging chamber and they are ready for microscope stage!
Maximum mobility is usually reached in 15-20 minutes, since QDs need to penetrate outer actin level (see paper for details).
Next well
Next well
For the next well with cells use another (fresh) vial of QD/nanobody mix.