The Micronucleus Assay on Cryopreserved Whole Blood

For this study, blood samples were collected by venipuncture from 30 healthy donors aged between 17 and 65 years. MN data from 20 donors were reused from the paper of Beyls et al.⁸ Collection of the blood samples is in accordance with the guidelines of the Ethics Committee of Ghent University Hospital (registration number:2019/1565), Belgium. Written informed consent was obtained from all the participants. See the Table of Materials for details related to all materials and reagents used in this protocol.

1. Collection of blood samples

1. Before starting, ensure that written informed consent of the participants is available and ethical guidelines are followed properly .
2. Collect blood into Li-Heparin tubes and store at room temperature until ready for processing for cryopreservation.

2. Cryopreservation of whole blood

NOTE: All the steps until cell harvesting are performed aseptically in sterile laminar airflow.

1. To cryopreserve 1 mL of blood, make 1 mL of freezing medium by mixing 800 µL of Fetal Calf Serum (FCS) and 200 µL of dimethyl sulfoxide (DMSO).
   NOTE: The freezing medium is prepared in a volume equal to the blood samples that need to be frozen.
2. For cryopreservation of whole blood, transfer the blood sample from the heparinized tube to 15 or 50 mL centrifuge tubes
   NOTE: The capacity of the tube depends on the volume of the blood.
3. Vortex the blood at low speed with continuous but dropwise addition of an equal volume of freezing medium.
4. Transfer 2 mL of blood-freeze mix into cryovials (2 mL) where each aliquot has 1 mL of blood sample mixed with 1 mL of freezing mixture.
5. Stepwise gradual freezing
   1. Transfer cryovials to a cryobox containing isopropanol and put in a freezer (-80 °C) for overnight.
   2. Transfer the cryovials to liquid nitrogen for long-term use.

3. Thawing of cryopreserved blood

NOTE: To limit the total time of the thawing process, a maximum of 8 cryovials (2 mL each) should be handled at a time.

1. Preparation
   1. Prewarm 200 mL of sterile Phosphate-buffered Saline (PBS) to 37 °C before thawing the blood.
   2. Prepare a working solution of the required culture medium under sterile conditions. Ensure that medium per culture includes 1.6 mL of complete medium: Rosewell Park Memorial Institute (RPMI) media supplemented with penicillin/streptomycin (0.5%), 0.2 mL of FCS, 20 µL of sodium pyruvate, and 2 µL of β-mercaptoethanol.
      NOTE: Since a significant number of blood cells can be lost while thawing, the thawed blood is cultured in small wells like 24-well plates with a total volume of 2 mL. One multi-well plate per dose is used.
   3. For each culture well, indicate the donor code and A/B on the lid along with the date and dose, as suggested below (Figure 1).

Figure 1: Suggested layout of a 24-well plate to culture whole blood for the micronucleus assay. Mark the wells and indicate the donor codes at appropriate places on the lid. For each patient sample, duplicate wells should be adjacent to each other. Note that it is a suggestive layout for a 10 x 10 collimator and can be changed according to the size of the collimator or samples. Please click here to view a larger version of this figure.

2. Steps for thawing and culturing whole blood
   1. Warm the water bath to 37 °C.
   2. Take out the required cryovials from the liquid nitrogen (the number depends on the requirements such as the number of doses).
      ​NOTE: One cryovial contains 1 mL of blood and 1 mL of freezing medium. Therefore, two cultures can be set up with blood from one cryovial.
   3. Partially thaw the cryovials in the warm water bath (until small ice crystals are still seen).
   4. Take out the vials from the water bath and clean the outside of the vials with sterile tissue paper.
   5. In a sterile laminar airflow hood , open the vials very slowly to prevent air bubbles formed inside the tubes from spilling out.
   6. Resuspend the contents of each vial and individually transfer them to a 15 mL conical centrifuge tube (1 tube per vial).
   7. To the respective tubes, add dropwise 1 mL of warm (37 °C) PBS while gently swirling the tube. Resuspend with a P1000 pipette.
   8. For dilution and uniform washing, add dropwise 7 mL of prewarmed PBS (37 °C) and resuspend with a P1000 pipette.
   9. Centrifuge the blood for 8 min at 180 × g (with setting at acceleration 1, brake 1) at room temperature.
   10. During this waiting step, fill the empty wells (other than the one where cells will be cultured) with 500 µL of PBS.
   11. Place the well plates back in the incubator to prewarm.
   12. Once the centrifugation is completed, carefully remove the supernatant, leaving behind approximately 1 mL to avoid disturbing the cell pellet.
   13. Resuspend the pellet in the remaining supernatant with a P1000 pipette.
   14. Add dropwise 8 mL of warm PBS (37 °C) into the respective tubes while gently swirling the tubes. Afterwards, resuspend with a P1000 pipette.
   15. Centrifuge the resuspended blood for 8 min at 180 × g (with setting at acceleration 9, brake 9).
   16. During this waiting step, transfer 200 µL of culture medium to each of the marked wells.
   17. Keep the plates back in the incubator to remain at 37 °C.
   18. After centrifugation, remove the supernatant in a continuous motion (to avoid disturbing the loose cell pellet), but leave behind approximately 80 µL of the supernatant.
   19. To the pellet, add 280 µL of FCS (room temperature) and resuspend (to a total volume of 360 µL).
   20. Transfer 180 µL of the cell suspension to each well and resuspend (= 0.5 mL of "blood"/culture). The total volume in each well is now 380 µL, resulting in a 2 mm medium layer.
   21. Incubate the plates in a CO₂ incubator at 37 °C for at least 10 min before irradiation.

4. G0 MN assay

1. Take out the plates from the incubator and irradiate the cell culture with required doses such as 0.5, 1, and 2 Gy X-rays (220 kV, 13 mA, 0.15 mm Cu) at room temperature. Use sham irradiated samples as controls to identify spontaneous MN yields.
2. Immediately after irradiation, add 1.62 mL of culture medium (37 °C) to each well.
3. To stimulate cell division in T-lymphocytes, add 40 µL of Phytohemagglutinin (PHA) to each well and resuspend thoroughly.
4. Place these plates back in the incubator (5% CO₂, 37 °C).
   NOTE: This will be the starting time of the assay.
5. Block the cytokinesis 23 h post stimulation by adding 8 µL of cytochalasin B (6 µg/mL) per well and resuspend properly.
6. Harvest the cell cultures 70 h post stimulation/culture time. Resuspend and transfer the cells to a 15 mL tube. Rinse each well with 2 mL of PBS and add this PBS to the respective 15 mL tube.
7. Centrifuge the tubes for 8 min at 180 × g, at room temperature.
8. Discard the supernatant but leave approximately 500 µL over the pellet.
9. Vortex the pellet (at full speed) and while vortexing, slowly add dropwise 2 mL of cold potassium chloride (KCl, 4 °C).
10. Immediately centrifuge the cells at 180 × g for 8 min.
11. Discard the supernatant, leaving approximately 500 µL of the supernatant.
12. Vortex the pellet (at full speed) and while vortexing, slowly add dropwise 2 mL of cold fixative 1 (Methanol/Acetic acid/Ringer solution in a ratio of 4:1:5).
13. Leave the sample tubes overnight at 4 °C (minimum 12 h and maximum 96 h).
14. Centrifuge for 8 min at 180 × g.
15. Discard the supernatant.
16. Vortex the pellet (at full speed) and while vortexing, slowly add dropwise 2 mL of cold fixative 2 (methanol/acetic acid in a ratio of 4:1).
17. Centrifuge for 8 min at 180 × g.
18. Discard the supernatant.
19. Vortex the pellet (at full speed) and then add slowly (dropwise), while vortexing, 2 mL of cold fixative 2 to the pellet.
20. Leave the sample tubes at 4 °C (minimum 12 h and maximum 96 h).
21. Clean the slides with isopropanol and label them properly.
    NOTE: Use printed stickers to label as marker ink is easily wiped off while processing.
22. Centrifuge the sample tubes for 8 min at 180 × g.
23. Transfer the supernatant to another tube to concentrate the cells according to the size of the pellet.
24. Vortex the pellet.
25. Drop 40 µL of the fixed cells on a dry and clean slide.
26. Let the slides dry at room temperature. Store the slides in a box or stain immediately for observation.

5. Acridine orange (AO) staining

1. Immerse the slides in AO stain for 1 min, followed by a quick wash in the distilled water, and then, place the slides in phosphate buffer (pH 6.8) for 1 min.
2. Take out the slides from the buffer solution and with clean tissue paper, dry the back of the slides, and put the slides on a clean tissue paper.
3. Drop 20 µL of the phosphate buffer on top of the slide and gently cover it with a clean coverslip, avoiding air bubbles.
4. Seal these slides with silicone cement.
   ​NOTE: Since AO is light-sensitive and fades over time, for a nice contrast and fluorescence, scoring of the slides is recommended within 5 days of staining. Always store slides in a cold room (4 °C), when not examined.

6. Scoring stained slides

1. Place your slide under a fluorescence microscope and examine 1,000 binucleated cells (BNs). Manually count MNs, which are one of the six biomarkers of toxicity. To do so, have two independent scorers examine 500 BN cells/slide under the same magnification.
   NOTE: Following are some highlights of the scoring criteria, as recommended by Fenech to score the stained slides⁹
   1. Select a BN cell by looking for nicely rounded cells with intact cytoplasm, with two well-distinguished nuclei of approximately the same size, regular shape, and staining pattern. Ensure that the cytoplasm of the BN cell is distinguishable from the cytoplasm of the adjacent cell.
   2. Score a MN, noting that it is morphologically identical to but smaller than the main nuclei; even the largest MN cannot be more than 1/3 of the diameter of the main nuclei. Score a MN only if it does not touch the main nuclei or at least the micronuclear boundary is distinguishable from the boundary of the main nuclei.