Ex vivo Analysis of DNA Repair Capacity of Human Peripheral Blood Mononuclear Cells by a Modified Host Cell Reactivation Assay

Materials and Reagents

1. Centrifuge tubes 15, 50 ml (SARSTEDT, catalog numbers: 62.554.001, 62.547.254)
2. 1.5 ml reaction tube (SARSTEDT, catalog number: 72.706)
3. Petri dishes (SARSTEDT, catalog number: 83.3902)
4. Pipette tips 10 μl, 20 μl, 200 μl, 1,000 μl (SARSTEDT, catalog numbers: 70.1130, 70.116, 70.760.002, 70.762)
5. Serological pipettes 5 ml and 10 ml (SARSTEDT, catalog numbers: 86.1253.001 and 86.1254.001)
6. Leucosep^TM, with porous barrier, 50 ml, pre filled with Leucosep^TM separation medium (Greiner Bio-One GmbH, catalog number: 227288)
7. Sterile filter 0.2 μm (SARSTEDT, catalog number: 83.1826.001)
8. Cell culture plate, 6-well, Cell+ (SARSTEDT, catalog number: 83.3920.300)
9. 0.4 cm electroporation cuvettes (Molecular BioProducts, Inc., purchased via Thermo Fisher Scientific GmbH, catalog number: 5540-11)
10. FACS tubes 5 ml and 0.5 ml (SARSTEDT, catalog numbers: 55.1579, 55.673)
11. Plasmid-DNA solution pEGFP-N1 (Clontech, catalog number: 6085-1) and pDsRedExpress-N1 (Clontech, catalog number: 632429), each 900 ng/µl in Qiagen EB buffer (storage temperature for longer periods: -80 °C, otherwise keep at 4 °C)
12. Human venous blood, anticoagulated
13. KCl (Carl Roth, catalog number: 6781.3)
14. KH₂PO₄ (Carl Roth, catalog number: 3904.2)
15. Na₂HPO₄·12H₂O (Carl Roth, catalog number: N350.1)
16. NaCl (Carl Roth, catalog number: 3957.3)
17. S-Monovette^® K3 EDTA 4.9 ml and 7.5 ml (SARSTEDT, catalog numbers: 04.1931.001 and 01.1605.001)
18. RPMI 1640 without Phenol Red (Gibco^® by Life Technologies, purchased via Fisher Scientific GmbH, catalog number: 11835-063, storage temp. 4 °C)
19. Fetal Bovine Serum (Gibco^® by Life Technologies, chased via Fisher Scientific GmbH, catalog number: 10270-106)
20. EB Buffer, part of the QIAGEN Plasmid Plus Giga Kit (Qiagen GmbH, Hilden, catalog number: 12991)
21. BD FACS Sheath Solution with Surfactant (BD Biosciences, catalog number: 336911)
22. 1x PBS (see Recipes)
23. 0.9% NaCl solution (see Recipes)
24. PBMC medium (see Recipes)
    

Equipment

1. Pipettes, Eppendorf Research^® Plus 10 μl, 20 μl, 200 μl, 1,000 μl (Eppendorf, catalog numbers: 3123000020, 3123000039, 3123000055, 3123000063)
2. Stratalinker^® UV Crosslinker (Stratagene, catalog number: 400072, model: Model 1800)
3. Centrifuge with swinging bucket rotor (Eppendorf, catalog numbers: 5805000010, 5804709004)
4. Neubauer counting chamber improved (Carl Roth, catalog number: PC72.1)
5. Gene Pulser II with Capacitance extender plus and Pulse controller plus (Bio-Rad Laboratories, Inc., catalog numbers: 165-2106, 165-2108, 165-2110)
6. CO₂ Incubator Hera cell 150 (Kendro Laboratory Products, catalog number: 51013568)
7. BD FACSCalibur Flow Cytometry System with 488 nm laser (Becton, Dickinson and Company)
8. -80 °C freezer HFU600TV (Thermo Fisher Scientific^TM, catalog number: 11670823)
   

Software

1. CellQuest Pro (Becton, Dickinson and Company, version 4.0.2)
2. Prism (GraphPad Software, Inc.)
   

Procedure

Day 1

1. Prepare plasmid solutions
   1. pDsRedExpress-N1: Adjust concentration to a maximum of 900 ng/µl using EB buffer.
      Note: The higher the plasmid concentration is, the more likely plasmids lie above each other during irradiation and irradiation becomes less effective. This results in higher DNA repair capacities in the end. Irradiate half of the volume by pipetting droplets of 40 µl into a Petri dish (10 cm diameter). Irradiate with 5 kJ/m² UVC (254 nm) without lid of the Petri dish using Stratalinker^® UV Crosslinker. After irradiation, collect droplets in a 1.5 ml reaction tube. The other half of the plasmid solution remains non-irradiated.
   2. pEGFP-N1 needs no further treatment as it serves as transfection control. The concentration of plasmid solution should be > 600 ng/µl in order to avoid too high volumes. The ratio of absorbance at 260 nm and 280 nm should be ~1.8.
   3. For the measurement of DNA repair, mix 10 µg of pEGFP-N1 and 30 µg of irradiated pDsRedExpress-N1. For calculation of the correction factor, mix 10 µg of pEGFP-N1 and 30 µg of non-irradiated pDsRedExpress-N1 for each electroporation. Note that the volumes needed depend on the concentration of the plasmid solutions. Adjust the volume of the mixture to 100 µl using EB buffer. Prepare enough plasmid-DNA mixtures for the entire study planned. Store mixtures at 4 °C for short-term storage or at -80 °C for longer periods.
2. Isolate PBMCs from 12 ml of human blood using Leucosep^TM tubes according to the manufacturer’s instructions as follows:
   
   1. Dilute 12 ml anticoagulated blood 1:2 using 0.9% NaCl solution (24 ml final volume) and transfer into Leucosep^TM tubes.
   2. Centrifugate at 1,000 x g for 10 min in a swinging bucket rotor and switch off brakes. Figure 1A shows the Leucosep^TM tube before centrifugation and Figure 1B shows the tube after centrifugation.
      
      
      Figure 1. Leucosep^TM tube before and after centrifugation. A. Leucosep^TM tube filled with anticoagulated blood, which was previously diluted 1:2 with 0.9% NaCl solution. B. Leucosep^TM tube after density gradient centrifugation. The different layer can be seen clearly from top to bottom: plasma, enriched cells, separation medium, the porous barrier, again separation medium and finally erythrocytes and granulocytes.
      
      
   3. Pipet approximately 10 ml of plasma from the top in order to reach the enriched cell fraction more easily.
   4. Harvest the enriched cell fraction using a 1,000 µl Eppendorf pipette and transfer cells into a 50 ml centrifuge tube.
   5. Wash cells with 10 ml PBS and centrifuge at 250 x g for 10 min.
   6. Repeat the washing step two times each with 5 ml PBS. When adding 5 ml for the second time, count cells using a Neubauer counting chamber.
3. For each electroporation, fill 3 ml PBMC medium per well into a 6-well cell culture plate.
4. Centrifuge 2 x 10⁶ cells at 250 x g for 10 min in a 15 ml centrifuge tube for each electroporation. Prepare two tubes in order to be able to calculate DNA repair capacity of one sample properly. One tube will contain the irradiated pDsRedExpress-N1 and pEGFP-N1 and the other one the non-irradiated pDsRedExpress-N1 and pEGFP-N1. Resuspend pellet in 150 µl RPMI media without phenol red.
5. Add 100 µl of the previously prepared plasmid mixture to the cell suspension, so that the suspension contains 10 µg pEGFP-N1 and 30 µg pDsRedExpress-N1 (either irradiated or non-irradiated).
6. Transfer 250 µl of the cell suspension to electroporation cuvettes with 4 mm gap, place it into the shocking chamber of Gene Pulser II and start electroporation at 400 V and 500 µF.
   Note: Always electroporate one irradiated batch and one non-irradiated batch directly one after the other. These two will be used to calculate the DNA repair capacity of one sample.
7. Transfer electroporation batches in the order of electroporation into the prepared 6-well cell culture plate containing RPMI medium.
8. Incubate for 24 h at 37 °C, 5 % CO₂ and relative humidity of 95%.
   
   
   

9. Prepare one 1.5 ml reaction tube for each electroporation approach.
10. Mix cells in the wells of the cell culture plate using a 1,000 µl Eppendorf pipette before transferring 1.5 ml of the cell suspension into the 1.5 ml reaction tubes prepared previously. 
11. Centrifuge at 250 x g for 10 min. 
12. Remove 1.4 ml of the supernatant, resuspend cells in the remaining 100 µl, and transfer into 0.5 ml FACS tubes.
13. Place the 0.5 ml tube into a 5 ml tube and carry out FACS analysis using 488 nm laser.

Data analysis

FACS analysis was carried out using FACSCalibur, CellQuest Pro software and the 488 nm laser. Gating of the cells of interest (lymphocytes) was carried out with the control sample. For analysis of the sample containing the irradiated pDsRedExpress-N1 the same gates were used. Gating of the transfected control samples and the samples containing the irradiated pDsRedExpress-N1 are shown in Figures 2 and 3 respectively.


Figure 2. FACS analysis of the control sample. A. Control sample containing the non-irradiated pDsRedExpress-N1 with gated lymphocytes. B. Transfected cells gated with gate R1. The area lower right (R2) displays cells expressing only GFP, the area upper left (R3) displays cells expressing only DsRed and the area upper right (R4) displays cells expressing GFP as well as DsRed.


Figure 3. FACS analysis of the samples containing the irradiated pDsRedExpress-N1. A. Sample containing irradiated pDsRedExpress-N1 with gated lymphocytes. B. Transfected cells gated with gate R1. The area lower right (R2) displays cells expressing only GFP, the area upper left (R3) displays cells expressing only expressing DsRed and the area upper right (R4) displays cells expressing GFP as well as DsRed.

For calculation of DNA repair capacity, percentages of cells expressing GFP and DsRed are added up as follows. All GFP-expressing cells are the cells in gates R2 and R4. All DsRed-expressing cells are the cells in gates R3 and R4.
  For the correction of repair capacity with regard to efficacy of transfection, the correction factor F is needed. F is always calculated from the percentages of GFP and DsRed of the control sample. Hence, a control is needed for each transfection with irradiated pDsRedExpress-N1. F is the ratio of percentages of GFP-expressing cells to the DsRed-expressing cells of the control sample. DNA repair capacity is calculated according to Matt et al. (2016) as follows:



For statistical analyses, any version of Prism (GraphPad Software, Inc.) can be used. We recommend triplicates for the calculation of DNA repair capacity.

Notes

1. Pay attention to the time constants during electroporation. In our experiments, time constants are usually between 20 and 25 ms. Furthermore, too high time constants resulted in low transfection efficiencies or cell death.
2. To allow the laser to warm up, switch on flow cytometer approximately 30 min before FACS analysis.
3. To compensate for the non-optimal excitation of DsRed by the 488 nm laser, three times the amount of DsRed compared to GFP is used for electroporation. 
4. We recommend triplicates for each sample (three control approaches and three matching approaches containing the irradiated pDsRedExpress-N1).
   

Recipes

1. 1x PBS
   8 g NaCl
   0.20 g KCl
   2.88 g Na₂HPO₄·12H₂O
   1.24 g KH₂PO₄
   Add 1 L ddH₂O, pH 7.4
   
2. 0.9% NaCl solution
   9 g NaCl
   Add 1 L ddH₂O, sterile filter solution using 0.2 µm sterile filter
3. PBMC medium
   RPMI 1640 without phenol red
   20% fetal bovine serum
   Store at 4 °C
   

Acknowledgments

The authors thank Dr. med. Karin Rupprecht (Center for Traditional Chinese Medicine–TCM Sigmaringen) and Dr. med. Adrian Schulte (F. X. Mayr Bodensee Center Überlingen) for drawing of the blood samples and informing of the volunteers.

Competing interests

One of the authors has consulting contracts with MSE Pharmazeutika GmbH, Bad Homburg, Germany.

Ethics

All experiments were conducted in accordance with the declaration of Helsinki as well as approved by the ethics committee of the Landesärztekammer Baden-Württemberg, Germany. The volunteers were informed before collection of the samples and gave their written consent to the use of their blood samples.

References

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