The authors would like to thank L. Pieters, T. Thiron, and G. De Smet for their technical support. We are thankful to all the volunteers who donated blood for the study. The work was financially supported by the Research Foundation- Flanders (FWO) under Grant (T000118N).

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.&#160;Collection of blood samples
<ol>
	<li>Before starting, ensure that written informed consent of the participants is available and ethical guidelines are followed properly .</li>
	<li>Collect blood into Li-Heparin tubes and store at room temperature until ready for processing for cryopreservation.</li>
</ol>
2.&#160;Cryopreservation of whole blood
NOTE: All the steps until cell harvesting are performed aseptically in sterile laminar airflow.
<ol>
	<li>To cryopreserve 1 mL of blood, make 1 mL of freezing medium by mixing 800 &#181;L of Fetal Calf Serum (FCS) and 200 &#181;L of dimethyl sulfoxide (DMSO). 
	NOTE: The freezing medium is prepared in a volume equal to the blood samples that need to be frozen.</li>
	<li>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.</li>
	<li>Vortex the blood at low speed with continuous but dropwise addition of an equal volume of freezing medium.</li>
	<li>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.</li>
	<li>Stepwise gradual freezing
	<ol>
		<li>Transfer cryovials to a cryobox containing isopropanol and put in a&#160;freezer&#160;(-80 &#176;C) for overnight.</li>
		<li>Transfer the cryovials to liquid nitrogen for long-term use.</li>
	</ol>
	</li>
</ol>
3.&#160;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.
<ol>
	<li>Preparation
	<ol>
		<li>Prewarm 200 mL of sterile Phosphate-buffered Saline (PBS) to 37 &#176;C before thawing the blood.</li>
		<li>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 &#181;L of sodium pyruvate, and 2 &#181;L of &#946;-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.</li>
		<li>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).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65855/65855fig01.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65855/65855fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Steps for thawing and culturing whole blood
	<ol>
		<li>Warm the water bath to 37 &#176;C.</li>
		<li>Take out the required cryovials from the liquid nitrogen (the number depends on the requirements such as the number of doses). 
		&#8203;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.</li>
		<li>Partially thaw the cryovials in the warm water bath (until small ice crystals are still seen).</li>
		<li>Take out the vials from the water bath and clean the outside of the vials with sterile tissue paper.</li>
		<li>In a sterile laminar airflow hood , open the vials very slowly to prevent air bubbles formed inside the tubes from spilling out.</li>
		<li>Resuspend the contents of each vial and individually transfer them to a 15 mL conical centrifuge tube (1 tube per vial).</li>
		<li>To the respective tubes, add dropwise 1 mL of warm (37 &#176;C) PBS while gently swirling the tube. Resuspend with a P1000 pipette.</li>
		<li>For dilution and uniform washing, add dropwise 7 mL of prewarmed PBS (37 &#176;C) and resuspend with a P1000 pipette.</li>
		<li>Centrifuge the blood for 8 min at 180 &#215; g (with setting at acceleration 1, brake 1) at room temperature.</li>
		<li>During this waiting step, fill the empty wells (other than the one where cells will be cultured) with 500 &#181;L of PBS.</li>
		<li>Place the well plates back in the incubator to prewarm.</li>
		<li>Once the centrifugation is completed, carefully remove the supernatant, leaving behind approximately 1 mL to avoid disturbing the cell pellet.</li>
		<li>Resuspend the pellet in the remaining supernatant with a P1000 pipette.</li>
		<li>Add dropwise 8 mL of warm PBS (37 &#176;C) into the respective tubes while gently swirling the tubes. Afterwards, resuspend with a P1000 pipette.</li>
		<li>Centrifuge the resuspended blood for 8 min at 180 &#215; g (with setting at acceleration 9, brake 9).</li>
		<li>During this waiting step, transfer 200 &#181;L of culture medium to each of the marked wells.</li>
		<li>Keep the plates back in the incubator to remain at 37 &#176;C.</li>
		<li>After centrifugation, remove the supernatant in a continuous motion (to avoid disturbing the loose cell pellet), but leave behind approximately 80 &#181;L of the supernatant.</li>
		<li>To the pellet, add 280 &#181;L of FCS (room temperature) and resuspend (to a total volume of 360 &#181;L).</li>
		<li>Transfer 180 &#181;L of the cell suspension to each well and resuspend (= 0.5 mL of &#34;blood&#34;/culture). The total volume in each well is now 380 &#181;L, resulting in a 2 mm medium layer.</li>
		<li>Incubate the plates in a CO2 incubator at 37 &#176;C for at least 10 min before irradiation.</li>
	</ol>
	</li>
</ol>
4.&#160;G0 MN assay
<ol>
	<li>Take out the plates from the incubator and irradiate the cell culture with required doses such as 0.5,&#160;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.</li>
	<li>Immediately after irradiation, add 1.62 mL of culture medium (37 &#176;C) to each well.</li>
	<li>To stimulate cell division in T-lymphocytes, add 40 &#181;L of Phytohemagglutinin (PHA) to each well and resuspend thoroughly.</li>
	<li>Place these plates back in the incubator (5% CO2, 37 &#176;C). 
	NOTE: This will be the starting time of the assay.</li>
	<li>Block the cytokinesis 23 h post stimulation by adding 8 &#181;L of cytochalasin B (6 &#181;g/mL) per well and resuspend properly.</li>
	<li>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.</li>
	<li>Centrifuge the tubes for 8 min at 180 &#215; g, at room temperature.</li>
	<li>Discard the supernatant but leave approximately 500 &#181;L over the pellet.</li>
	<li>Vortex the pellet (at full speed) and while vortexing, slowly add dropwise 2 mL of cold potassium chloride (KCl, 4 &#176;C).</li>
	<li>Immediately centrifuge the cells at 180 &#215; g for 8 min.</li>
	<li>Discard the supernatant, leaving approximately 500 &#181;L of the supernatant.</li>
	<li>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).</li>
	<li>Leave the sample tubes overnight at 4 &#176;C (minimum 12 h and maximum 96 h).</li>
	<li>Centrifuge for 8 min at 180 &#215; g.</li>
	<li>Discard the supernatant.</li>
	<li>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).</li>
	<li>Centrifuge for 8 min at 180 &#215; g.</li>
	<li>Discard the supernatant.</li>
	<li>Vortex the pellet (at full speed) and then add slowly (dropwise), while vortexing, 2 mL of cold fixative 2 to the pellet.</li>
	<li>Leave the sample tubes at 4 &#176;C (minimum 12 h and maximum 96 h).</li>
	<li>Clean the slides with isopropanol and label them properly. 
	NOTE: Use printed stickers to label as marker ink is easily wiped off while processing.</li>
	<li>Centrifuge the sample tubes for 8 min at 180 &#215; g.</li>
	<li>Transfer the supernatant to another tube to concentrate the cells according to the size of the pellet.</li>
	<li>Vortex the pellet.</li>
	<li>Drop 40 &#181;L of the fixed cells on a dry and clean slide.</li>
	<li>Let the slides dry at room temperature. Store the slides in a box or stain immediately for observation.</li>
</ol>
5.&#160;Acridine orange (AO) staining
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Drop 20 &#181;L of the phosphate buffer on top of the slide and gently cover it with a clean coverslip, avoiding air bubbles.</li>
	<li>Seal these slides with silicone cement. 
	&#8203;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 &#176;C), when not examined.</li>
</ol>
6.&#160;Scoring stained slides
<ol>
	<li>Place your slide under a fluorescence&#160;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&#160;stained slides9
	<ol>
		<li>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.</li>
		<li>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&#160;of the diameter of the main nuclei. Score a&#160;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.</li>
	</ol>
	</li>
</ol>

Examining Micronuclei Yields in Irradiated Whole Blood Samples

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J., Camps, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+chromosomal+aberrations+in+solid+tumors.">Patterns of chromosomal aberrations in solid tumors.</a> Recent Results in Cancer Research. 200, 115-142 (2015).</li><li>Jiao, Y., Cao, F., Liu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation-induced+cell+death+and+its+mechanisms.">Radiation-induced cell death and its mechanisms.</a> Health Physics. 123 (5), 376-386 (2022).</li><li>Roederer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+architecture+of+the+human+immune+system:+a+bioresource+for+autoimmunity+and+disease+pathogenesis.">The genetic architecture of the human immune system: a bioresource for autoimmunity and disease pathogenesis.</a> Cell. 161 (2), 387-403 (2015).</li><li>Bankoglu, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cryopreservation+on+DNA+damage+and+DNA+repair+activity+in+human+blood+samples+in+the+comet+assay.">Effect of cryopreservation on DNA damage and DNA repair activity in human blood samples in the comet assay.</a> Archives of Toxicology. 95, 1831-1841 (2021).</li><li>Zijno, A., Saini, F., Crebelli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suitability+of+cryopreserved+isolated+lymphocytes+for+the+analysis+of+micronuclei+with+the+cytokinesis-block+method.">Suitability of cryopreserved isolated lymphocytes for the analysis of micronuclei with the cytokinesis-block method.</a> Mutagenesis. 22, 311-315 (2007).</li><li>Sioen, S., Cloet, K., Vral, A., Baeyens, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cytokinesis-block+micronucleus+assay+on+human+isolated+fresh+and+cryopreserved+peripheral+blood+mononuclear+cells.">The cytokinesis-block micronucleus assay on human isolated fresh and cryopreserved peripheral blood mononuclear cells.</a> Journal of Personalized Medicine. 10 (3), 125 (2020).</li><li>Cheng, L., Wang, L. E., Spitz, M. R., Wei, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreserving+whole+blood+for+functional+assays+using+viable+lymphocytes+in+molecular+epidemiology+studies.">Cryopreserving whole blood for functional assays using viable lymphocytes in molecular epidemiology studies.</a> Cancer Letters. 166 (2), 155-163 (2001).</li><li>Beyls, E., Baeyens, A., Vral, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cytokinesis-block+micronucleus+assay+for+cryopreserved+whole+blood.">The cytokinesis-block micronucleus assay for cryopreserved whole blood.</a> International Journal of Radiation Biology. 97 (9), 1252-1260 (2021).</li><li>Fenech, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokinesis-block+micronucleus+cytome+assay.">Cytokinesis-block micronucleus cytome assay.</a> Nature Protocols. 2 (5), 1084-1104 (2007).</li><li>Ramachandran, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+thawing+of+cryopreserved+peripheral+blood+mononuclear+cells+for+use+in+high-throughput+human+immune+monitoring+studies.">Optimal thawing of cryopreserved peripheral blood mononuclear cells for use in high-throughput human immune monitoring studies.</a> Cells. 1 (3), 313-324 (2012).</li><li>H&#248;nge, B. L., Petersen, M. S., Olesen, R., M&#248;ller, B. 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The authors have no conflicts of interest to declare.

The modified protocol for the application of the CBMN assay is a relatively easy and convenient way to store blood samples in bulk. The procedure outlines all the minute but important details that need to be taken care of during cryopreservation and the CBMN assay. Other lab protocols normally use 10% DMSO in the freezing mixture, while our freezing mixture contains 20% DMSO along with 80% FCS7. As this freezing mixture is added in equal volumes to the whole blood sample, the final concentration is also 10% DMSO. To improve the cell survival rate and stimulate cell division, we add 1% sodium pyruvate and 0.1% beta-mercaptoethanol to the complete culture medium (cRPMI). This is in accordance with the cell-culture protocol established by our research group6,8. 
Although the problem of cell clumping while thawing could not be resolved completely in this protocol, cell segregation was better than the other conventional protocols. Instead of consistent, abrupt addition of culture media to recover cells post thawing, we obtained better results when prewarmed PBS (37 &#176;C) was added dropwise during the washing steps. This improvement helps in reducing cellular stress and minimizes clumping with a proven high recovery of cells. Furthermore, no clear difference in cell viability could be observed when PBS was used over RPMI. It has been validated by the group that the length of the cryopreservation period (up to 1 year) will not affect MN yields, both in irradiated and non-irradiated samples6,8. Studies suggest that good cell viability and proliferation of PBMCs can be achieved with gradual freezing at low temperatures (liquid nitrogen), followed by gradual addition of prewarmed medium while thawing10,11. Researchers have shown that the sub-populations of T-lymphocytes in thawed whole blood are comparable to those observed in thawed PBMCs12. Further, it is proven that cell subtypes recovered from cryopreserved whole blood samples are similar to those observed in fresh whole blood samples13,14. 
If we examine the indicative results obtained in the report, we see that the linear quadratic increase with dose (Figure 3) is in agreement with other literature reports on Linear energy transfer (LET)-induced micronuclei15,16. The variability in MN yields observed in the cryopreserved whole blood samples (Table 1) are in range with the one reported for fresh whole blood cultures8,17,18. The protocol described in detail here was validated on fresh and cryopreserved whole blood of 20 healthy volunteers8. The nuclear division index (NDI), which is an important parameter of cell proliferation observed in that report8 was in agreement with the one suggested for fresh whole blood19,20. Taken together, this optimized protocol of cryopreservation of whole blood and modified micronucleus assay provides a better cell yield and hence, adaptation of this protocol is recommended for radiosensitivity assessments. Since the reproducibility of the protocol has already been validated8, it is suggested to have applicability in large-scale and multi-center studies.

Ever since its discovery, the use of ionizing radiation (IR) has been a matter of debate among researchers because of its adverse effects on living beings. The detrimental effect is usually manifested by DNA damage such as double-stranded breaks (DSBs), and the failure to repair these DSBs leads to chromosomal aberrations and mutations, which are important hallmarks of cancer1,2. Such chromosomal aberrations can be examined by cytogenic assays such as the cytokinesis-block micronucleus (CBMN) assay. Micronuclei are lagging chromosomal fragments that cannot be incorporated into daughter nuclei and hence, are left behind during mitosis.
CBMN is a commonly used, reliable cytogenetic technique to assess chromosomal damage in individuals exposed to in vivo or in vitro ionizing radiation. Either fresh whole blood or isolated peripheral blood mononuclear cells (PBMCs) can be used in the CBMN assay. Fresh whole blood is mostly the biological material of choice since the isolation and processing of PBMCs can be time-consuming and is accompanied by a loss of serum plasma that acts as the supporting medium for cell survival and growth. To achieve a good binucleated cell yield, fresh whole blood should be processed immediately after collection. However, the need for immediate processing can be logistically challenging during time constraints. Moreover, when many samples are supposed to be acquired over an extended period or collected at points away from the processing centers, storage of fresh blood samples can be a limiting factor3,4.
Further, to allow repeated MN analysis in the same individual/patient, freezing of blood samples would be beneficial. One way to store lymphocytes for later application of the CBMN assay is by freezing isolated PBMCs5,6. This technique, however, requires several processing steps before the PBMCs can be frozen. Therefore, cryopreservation of whole blood would represent a simple and time-efficient alternative to the cryopreservation of isolated PBMCs. Little information is available concerning the use of frozen whole blood for cytogenetic assays or assays that require the proliferation of lymphocytes. Only one paper reports the use of cryopreserved whole blood for metaphase analysis7.
As cryopreservation of whole blood would offer many advantages in the field of biomonitoring, biodosimetry, and radiosensitivity assessment, our group optimized a cryopreservation protocol for whole blood that allows the application of the CBMN assay8. We demonstrated that lymphocytes present in cryopreserved whole blood cultures retain their genomic integrity and proliferation capacity for at least 1 year. In this methods paper, we describe in detail the cryopreservation procedure and CBMN assay protocol, which was optimized by Beyls et al.8, and report findings obtained for frozen blood samples of 30 healthy individuals. For the CBMN assay, the blood cultures were irradiated in vitro with doses of 0.5, 1, and 2 Gy to assess the MN response in lymphocytes of cryopreserved whole blood samples.

<table><tbody><tr><td>15 mL centrifuge tubes</td><td>Greiner</td><td>188271</td><td></td></tr><tr><td>24-well cell suspension plate&amp;nbsp;</td><td>VWR</td><td>734-2779</td><td></td></tr><tr><td>96% alcohol</td><td>ChemLab</td><td>CL00.1807.2500</td><td></td></tr><tr><td>Acetic acid</td><td>Merck life science</td><td>8,18,75,52,500</td><td></td></tr><tr><td>Acridine orange</td><td>Merck life science</td><td>235474-5g</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Merck life science</td><td>C5670-100g</td><td></td></tr><tr><td>cover slips</td><td>VWR</td><td>631-1365</td><td>22 x 50</td></tr><tr><td>Cryobox (Mr.Frosty)</td><td>Nalgene, Sigma Aldrich</td><td></td><td></td></tr><tr><td>Cryovials 2ml</td><td>Novolab</td><td>A04573</td><td></td></tr><tr><td>Cytochalsin B&amp;nbsp;</td><td>Merck life science</td><td>C6762-10</td><td>10 mg</td></tr><tr><td>Dimethyl sulphoxide (DMSO)</td><td>Merck life science</td><td>D4540-500ml</td><td></td></tr><tr><td>Fetal calf serum (FCS)</td><td>Thermo Fischer scientific</td><td>10270-106</td><td></td></tr><tr><td>Fixative 1</td><td></td><td></td><td>Methanol/acetic acid/ringer in a ratio of 4:1:5</td></tr><tr><td>Fixative 2</td><td></td><td></td><td>Methanol/acetic acid in a ratio of 4:1</td></tr><tr><td>GURR buffer</td><td>Thermo Fischer scientific</td><td>10582013</td><td>phosphate buffer (pH 6.8)</td></tr><tr><td>KCl</td><td>Merck life science</td><td>1,04,93,60,250</td><td>75 mM</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck life science</td><td>1,04,87,30,250</td><td></td></tr><tr><td>Li-heparin tubes</td><td>BD Life sciences</td><td>367526-LH170 I.U.</td><td>BD Vacutainer</td></tr><tr><td>Methanol</td><td>fisher scinetific</td><td>M/4000/17</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;2&lt;/sub&gt;</td><td>Merck life science</td><td>10,65,80,500</td><td></td></tr><tr><td>NaCl</td><td>Merck life science</td><td>S7653-1kg</td><td></td></tr><tr><td>Object slides</td><td>VWR</td><td>MENZAA00000112E04</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Fischer scientific</td><td>15140-122</td><td>10,000 U/mL + 10,000 &amp;micro;g/mL</td></tr><tr><td>Phytohemagglutinin (PHA-M)</td><td>Thermo Fischer scientific</td><td>10576-015</td><td></td></tr><tr><td>Ringer solution</td><td></td><td></td><td>contains NaCl, KCl, CaCl&lt;sub&gt;2 &lt;/sub&gt;dissolved in distilled water</td></tr><tr><td>RPMI-1640</td><td>Thermo Fischer scientific</td><td>52400041</td><td></td></tr><tr><td>Silicon rubber adhesive sealent</td><td>collall</td><td>CF-100</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Thermo Fischer scientific</td><td>11360039</td><td></td></tr><tr><td>Sterile warm PBS (37 &amp;deg;C)</td><td></td><td></td><td>contains NaCl,&amp;nbsp; Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;2&lt;/sub&gt;, KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; dissolved in distilled water</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Thermo Fischer scientific</td><td>31350-010</td><td></td></tr></tbody></table>

the micronucleus assay on cryopreserved whole blood

The in vitro cytokinesis-block micronucleus (CBMN) assay is a widely used technique in radiobiology research, biological dosimetry, genotoxicity studies, and in vitro radiosensitivity testing. This cytogenetic method is based on the detection of micronuclei in binucleated cells resulting from chromosomal fragments lagging during cell division. Fresh whole blood samples are the most preferred sample type for the CBMN assay. However, the disadvantages of working with fresh blood samples include immediate processing after blood collection and the limited number of repeated analyses that can be performed without extra blood sampling. 
As the need for fresh blood samples can be logistically challenging, CBMN assay on cryopreserved whole blood samples would be of great advantage, especially in large-scale patient studies. This paper describes a protocol to freeze whole blood samples and to perform the CBMN assay on these frozen blood samples. Blood samples from healthy volunteers have been frozen and thawed at different time points and then, subjected to a modified micronucleus assay protocol. The results demonstrate that this optimized procedure allows the performance of the CBMN assay on frozen blood samples. The described cryopreservation protocol may also be very useful for other cytogenetic assays and a variety of functional assays requiring proliferating lymphocytes.

To validate the reproducibility of the protocol, we performed the MN assay on cryopreserved blood samples of 30 healthy volunteers aged between 17 and 65 years. The mean age of the group is 35 years. The cryopreservation time ranged from 1 week to 154 weeks. After exposing the whole blood cell culture to different radiation doses (0.5, 1, and 2 Gy), the micronuclei yield in 1,000 BN cells was examined under a microscope. Nicely rounded binucleated cells (as can be seen in Figure 2) indicate a successful retrieval of healthy viable cells from cryopreserved whole blood samples. Of note, the radiation response of lymphocytes remained stable after long-term storage at ultra-low temperatures (liquid nitrogen). This observation is in line with the response expected from fresh blood samples.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65855/65855fig02v2.jpg" /> 
Figure 2: Micronuclei as observed in binucleated cells retrieved from a frozen whole blood sample that was cryopreserved 1 year ago. (A) Magnification 200x, (B) Magnification 400x; Arrows pointing to the MNs. Scale bar = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65855/65855fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
With increased doses of radiation (0.5, 1, and 2 Gy), a linear-quadratic increase in micronucleus yield was observed (Table 1 and Figure 3). The MN yields in sham irradiated control samples (0 Gy) represent the background MN yields that are mainly the result of lagging chromosomes.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dose (Gy)</td>
			<td>0</td>
			<td>0.5</td>
			<td>1</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Average</td>
			<td>18</td>
			<td>107</td>
			<td>247</td>
			<td>601</td>
		</tr>
		<tr>
			<td>SD</td>
			<td>10.8</td>
			<td>23.9</td>
			<td>53.3</td>
			<td>101.1</td>
		</tr>
		<tr>
			<td>CV (%)</td>
			<td>59.9</td>
			<td>22.3</td>
			<td>21.6</td>
			<td>16.8</td>
		</tr>
		<tr>
			<td>Range</td>
			<td>5-41&#160;&#160;</td>
			<td>62-140</td>
			<td>139-324</td>
			<td>395-755</td>
		</tr>
	</tbody>
</table>
Table 1: Range and coefficient of variation along with average MN yield, as observed in cryopreserved whole blood samples of 30 healthy donors, indicative of inter-individual variability. Abbreviations: CV = coefficient of variation; SD = standard deviation.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65855/65855fig03.jpg" /> 
Figure 3: Micronuclei yields as observed in control and irradiated cryopreserved whole blood samples of 30 donors. Cryopreservation periods ranged from 1 week to 154 weeks. Scatter plot graph shows&#160;individual values. Lines in clusters represent the Mean &#177; SD of the group. Abbreviations: MN = micronuclei; BN = binucleated. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65855/65855fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To investigate the interindividual variation in the MN yield of persons, the coefficient of variation (CV) was calculated (see Table 1). For radiation-induced MN, a CV &#60; 25% was obtained for all doses (0.5, 1, and 2 Gy), which indicates good reproducibility of the modified protocol.

Watch this Scientific Journal Video about Cryopreservation of Whole Blood Samples for High Throughput Analysis at JoVE.com

Author Spotlight: Cryopreservation of Whole Blood Samples for High Throughput Analysis 

Here we present an optimized protocol for the cytokinesis-block micronucleus assay on cryopreserved whole blood samples. This optimized method of cryopreservation of whole blood for micronucleus analysis is a reliable technique for large-scale sampling and multi-center studies and can be used for other blood-related assays as well.

The Micronucleus Assay on Cryopreserved Whole Blood

The in vitro cytokinesis-block micronucleus (CBMN) assay is a widely used technique in radiobiology research, biological dosimetry, genotoxicity studies, ...

the-micronucleus-assay-on-cryopreserved-whole-blood

Nanyang Technological University

Research

JoVE Journal

Cancer Research

3.6K Views.  Ghent University. Ons onderzoek richt zich vooral op het onderzoeken van de impact van ioniserende straling op gezonde weefsels. We bestuderen de chromosomale radiogevoeligheid van mensen met kanker en mensen met DNA-herstelstoornissen. Daarnaast zijn we betrokken bij biologische dissymmetrie, waarbij we de DNA-schade beoordelen die wordt veroorzaakt door blootstelling aan ioniserende straling.Om het onderzoek in ons vakgebied vooruit te helpen, gebruiken we geautomatiseerde microscopische systemen voor...

Video: Auteur in de schijnwerpers: cryopreservatie van volbloedmonsters voor analyse met hoge doorvoer 

Isolation of Nuclei for Multiome Sequencing in Solid Tumor Specimen

Optimized Nuclei Isolation from Fresh and Frozen Solid Tumor Specimens for Multiome Sequencing

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) data, represents a breakthrough in our ability to discern tumor cell heterogeneity-a primary focus of translational cancer research at this time. However, the quality of sequencing data acquired using this advanced modality is highly dependent on the quality of the input material. 
Digestion conditions need to be optimized to maximize cell yield without sacrificing quality. This is particularly challenging in the context of solid tumors with dense desmoplastic matrices that must be gently broken down for cell release. Freshly isolated cells from solid tumor tissue are more fragile than those isolated from cell lines. Additionally, as the cell types isolated are heterogeneous, conditions should be selected to support the total cell population. 
Finally, nuclear isolation conditions must be optimized based on these qualities in terms of lysis times and reagent types/ratios. In this article, we describe our experience with nuclear isolation for the 10x Genomics multiome sequencing platform from solid tumor specimens. We provide recommendations for tissue digestion, storage of single-cell suspensions (if desired), and nuclear isolation and assessment.

Author Spotlight: Enhancing Nuclei Isolation for Multiome Sequencing in Challenging Tumor Microenvironments

The protocol provides a reliable and optimized approach to the isolation of nuclei from solid tumor specimens for multiome sequencing using the 10x Genomics platform, including recommendations for tissue dissociation conditions, cryopreservation of single-cell suspensions, and assessment of isolated nuclei.

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) ...

Nuclei Isolation from Mouse Cardiac Progenitor Cells at Single-Cell Resolution

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene expression and chromatin state of individual cells allows the identification of the cell type and state. Single-cell sequencing approaches have revealed a number of important characteristics of cardiac progenitor cell heterogeneity. However, these methods are generally restricted to fresh tissue, which limits studies with diverse experimental conditions, as the fresh tissue must be processed at once in the same run to reduce the technical variability. Therefore, easy and flexible procedures to produce data from methods such as single-nucleus RNA sequencing (snRNA-seq) and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) are needed in this area. Here, we present a protocol to rapidly isolate nuclei for subsequent single-nuclei dual-omics (combined snRNA-seq and snATAC-seq). This method allows the isolation of nuclei from frozen samples of cardiac progenitor cells and can be combined with platforms that use microfluidic chambers.

Author Spotlight: Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution  

Here, we present a protocol describing cell nuclei preparation. After microdissection and enzymatic dissociation of cardiac tissue into single cells, the progenitor cells were frozen, followed by isolation of pure viable cells, which were used for single-nucleus RNA sequencing and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing analyses.

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene ...

Developmental Biology

High-Quality Nuclei from iPSC-Derived Hematopoietic Cells for Multiomics Studies

Nuclear Isolation from Cryopreserved In Vitro Derived Blood Cells

Induced pluripotent stem cell (iPSC)-based models are excellent platforms to understand blood development, and iPSC-derived blood cells have translational utility as clinical testing reagents and transfusable cell therapeutics. The advent and expansion of multiomics analysis, including but not limited to single nucleus RNA sequencing (snRNAseq) and Assay for Transposase-Accessible Chromatin sequencing (snATACseq), offers the potential to revolutionize our understanding of cell development. This includes developmental biology using in vitro hematopoietic models. However, it can be technically challenging to isolate intact nuclei from cultured or primary cells. Different cell types often require tailored nuclear preparations depending on cellular rigidity and content. These technical difficulties can limit data quality and act as a barrier to investigators interested in pursuing multiomics studies. Specimen cryopreservation is often necessary due to limitations with cell collection and/or processing, and frozen samples can present additional technical challenges for intact nuclear isolation. In this manuscript, we provide a detailed method to isolate high-quality nuclei from iPSC-derived cells at different stages of in vitro hematopoietic development for use in single-nucleus multiomics workflows. We have focused the method development on the isolation of nuclei from iPSC-derived adherent stromal/endothelial cells and non-adherent hematopoietic progenitor cells, as these represent very different cell types with regard to structural and cellular identity. The described troubleshooting steps limited nuclear clumping and debris, allowing the recovery of nuclei in sufficient quantity and quality for downstream analyses. Similar methods may be adapted to isolate nuclei from other cryopreserved cell types.

Author Spotlight: Advancing In Vitro Blood Cell Production with Single-Cell Multiomics and Functional Genomics

We describe a protocol for extracting high-quality nuclei from cryopreserved induced pluripotent stem cell-derived stromal/endothelial and blood cell types to support single-nucleus next-generation sequencing analyses. Producing high-quality, intact nuclei is imperative for multiomics experiments but can be a barrier to entry in the field for some laboratories.

Induced pluripotent stem cell (iPSC)-based models are excellent platforms to understand blood development, and iPSC-derived blood cells have translational ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Bioengineering

Automation of the Micronucleus Assay Using Imaging Flow Cytometry and Artificial Intelligence

The micronucleus (MN) assay is used worldwide by regulatory bodies to evaluate chemicals for genetic toxicity. The assay can be performed in two ways: by scoring MN in once-divided, cytokinesis-blocked binucleated cells or fully divided mononucleated cells. Historically, light microscopy has been the gold standard method to score the assay, but it is laborious and subjective. Flow cytometry has been used in recent years to score the assay, but is limited by the inability to visually confirm key aspects of cellular imagery. Imaging flow cytometry (IFC) combines high-throughput image capture and automated image analysis, and has been successfully applied to rapidly acquire imagery of and score all key events in the MN assay. Recently, it has been demonstrated that artificial intelligence (AI) methods based on convolutional neural networks can be used to score MN assay data acquired by IFC. This paper describes all steps to use AI software to create a deep learning model to score all key events and to apply this model to automatically score additional data. Results from the AI deep learning model compare well to manual microscopy, therefore enabling fully automated scoring of the MN assay by combining IFC and AI.

The micronucleus (MN) assay is a well-established test for quantifying DNA damage. However, scoring the assay using conventional techniques such as manual microscopy or feature-based image analysis is laborious and challenging. This paper describes the methodology to develop an artificial intelligence model to score the MN assay using imaging flow cytometry data.

The micronucleus (MN) assay is used worldwide by regulatory bodies to evaluate chemicals for genetic toxicity. The assay can be performed in two ways: by ...

PBMC Collection and Cryostorage for Mitochondrial Bioenergetics Analysis

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases and aging. Oxidative phosphorylation plays a decisive role, as it is crucial for the maintenance of energetic homeostasis. PBMCs have been identified as a minimally invasive sample to measure mitochondrial function and have been shown to reflect disease conditions. However, measurement of mitochondrial bioenergetic function can be limited by several factors in human samples. Limitations are the amount of samples taken, sampling time, which is often spread over several days, and locations. Cryopreservation of the collected samples can ensure consistent collection and measurement of samples. Care should be taken to ensure that the parameters measured are comparable between cryopreserved and freshly prepared cells. Here, we describe methods for isolating and cryopreserving PBMCs from human blood samples to analyze the bioenergetic function of the mitochondria in these cells. PBMC cryopreserved according to the protocol described here show only minor differences in cell number and viability, adenosine triphosphate levels, and measured respiratory chain activity compared with freshly harvested cells. Only 8-24 mL of human blood is needed for the described preparations, making it possible to collect samples during clinical studies multicentrally and determine their bioenergetics on site.

Author Spotlight: Preservation of Bioenergetic Parameters in Peripheral Blood Mononuclear Cells After Cryopreservation 

Isolated peripheral blood mononuclear cells can be used for the analysis of immune functions and disorders, metabolic diseases, or mitochondrial functions. In this work, we describe a standardized method for the preparation of PBMCs from whole blood and the subsequent cryopreservation. Cryopreservation makes this time and place independent.

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases ...

Immunology and Infection

Preparation of Peripheral Blood Mononuclear Cell Pellets and Plasma from a Single Blood Draw at Clinical Trial Sites for Biomarker Analysis

Analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials to establish proof of mechanism to evaluate effects of treatment, and help guide dose and schedule setting of therapeutics. From a single blood draw, peripheral blood mononuclear cells can be isolated and processed to analyze and quantify protein markers, and plasma samples can be used for the analysis of circulating tumor DNA, cytokines, and plasma metabolomics. Longitudinal samples from a treatment provide information on the evolution of a given protein marker, the mutational status and immunological landscape of the patient. This can only be achieved if the processing of the peripheral blood is carried out effectively in clinical sites and samples are properly preserved from the bedside to bench. Here, we present an optimized general-purpose protocol that can be implemented at clinical sites for obtaining PBMC pellets and plasma samples in multi-center clinical trials, that will enable clinical professionals in hospital laboratories to successfully provide high quality samples, regardless of their level of technical expertise. Alternative protocol variations are also presented that are optimized for more specific downstream analytical methods. We apply this protocol for studying protein biomarkers against DNA damage response (DDR) on X-ray irradiated blood to demonstrate the suitability of the approach in oncology settings where DDR drugs and/or radiotherapy have been practiced as well as in preclinical stages where mechanistic hypothesis testing is required.

This protocol details clinically implementable preparation of high quality PBMC and plasma biosamples at the clinical trial site that can be used for translational biomarker analysis.

Analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials to establish proof of mechanism to evaluate effects of ...

Medicine

Isolation and Cryopreservation of Highly Viable Human Peripheral Blood Mononuclear Cells From Whole Blood: A Guide for Beginners

Peripheral blood mononuclear cells (PBMCs) are commonly used in biomedical research on the immune system and its response to disease and pathogens. This detailed protocol describes the equipment, supplies, and steps for isolating, cryopreserving, and thawing high-quality and highly viable PBMCs from whole blood cells suitable for downstream applications such as flow cytometry and RNA-sequencing. Protocols for processing plasma and buffy coat from the whole blood in parallel and concurrently with PBMCs are also described. This easy-to-follow step-by-step protocol, which utilizes density gradient centrifugation to isolate PBMCs, is accompanied by a checklist of supplies, equipment, and preparation steps. This protocol is suitable for individuals with any prior experience with laboratory techniques and can be implemented in clinical or research laboratories. High-quality cell viability and RNA sequencing resulted from PBMCs collected by operators with no prior laboratory experience using this protocol.

This protocol presents an accessible guide for collecting, storing, and thawing peripheral blood mononuclear cells suitable for downstream analyses and workflows like flow cytometry and RNA sequencing. Plasma and buffy coat collections are also demonstrated.

Peripheral blood mononuclear cells (PBMCs) are commonly used in biomedical research on the immune system and its response to disease and pathogens. This ...

Extraction and Identification of Micronuclei from Human Peripheral Blood Lymphocytes

A micronucleus (MN) is an abnormal nuclear structure that forms in cells, particularly bone marrow or blood cells, when exposed to external damage, such as radiation, due to unresolved DNA damage or mitotic errors. Once formed, MNs can actively contribute to various carcinogenic processes, including inflammatory signaling and chromosomal genetic rearrangements. MNs contain nuclear DNA, histones, nucleoprotein fragments, and other active proteins, which are closely associated with their functions. Studying the formation and components of MNs is crucial for understanding their role in driving carcinogenic processes. The extraction and purification of MNs are essential to achieving these research objectives. However, the instability of the MN nuclear membrane and its susceptibility to rupture make these processes technically challenging. Currently, only a few studies have reported the use of density gradient centrifugation for MN separation. This study summarizes and simplifies the processes of MN separation and purification. Human peripheral blood lymphocytes exposed to radiation were isolated, and MNs were separated and purified using sucrose buffers of different concentrations in a two-step process. The integrity and purity of the MNs were verified, providing a clear and practical demonstration of the experimental procedure for researchers investigating the causes and functions of MNs.

This protocol outlines a method for extracting and purifying micronuclei from human lymphocytes using sucrose density gradient centrifugation. It provides an experimental basis for investigating the composition and function of micronuclei.

A micronucleus (MN) is an abnormal nuclear structure that forms in cells, particularly bone marrow or blood cells, when exposed to external damage, such ...

High-Quality Sample Preparation for Multiome 10X Genomics Analysis

High-Quality Brain and Bone Marrow Nuclei Preparation for Single Nuclei Multiome Assays

Single-cell analysis has become the approach of choice for unraveling the complexity of biological processes that require assessing the variability of individual cellular responses to treatment or infection with single-cell resolution. 
Many techniques for single-cell molecular profiling have been developed over the past 10 years, and several dedicated technologies have been commercialized. The 10X Genomics droplet-based single-cell profiling is a widespread technology that offers ready-to-use reagents for transcriptomic and multi-omic single-cell profiling. The technology includes workflows for single-cell and single-nuclei RNA sequencing (scRNA-Seq and snRNA-Seq, respectively), scATAC-Seq, single-cell immune profiling (BCR/TCR sequencing), and multiome. The latter combines transcriptional (scRNA-Seq) and epigenetic information (scATAC-Seq) coming from the same cell. 
The quality (viability, integrity, purity) of single-cell or single-nuclei suspensions isolated from tissues and analyzed by any of these approaches is critical for generating high-quality data. Therefore, the sample preparation protocols should be adapted to the particularities of each biological tissue and ensure the generation of high-quality cell and nuclei suspensions.
This article describes two protocols for preparing brain and bone marrow samples for the downstream multiome 10X Genomics pipeline. The protocols are performed stepwise and cover tissue dissociation, cell sorting, nuclei isolation, and quality control of prepared nuclei suspension that is used as starting material for cell partitioning and barcoding, library preparation, and sequencing. These standardized protocols produce high-quality nuclei libraries and robust and reliable data.

Author Spotlight: Advancing Biomedical Research Through Single Cell Analysis 

The success of single-cell/single-nuclei transcriptomics and multi-omics largely depends on the quality of cells/nuclei. Therefore, isolating cells/nuclei from tissue and their purification must be highly standardized. This protocol describes the preparation of nuclei from the brain and bone marrow for downstream single-nuclei multiome assay.

Single-cell analysis has become the approach of choice for unraveling the complexity of biological processes that require assessing the variability of ...

The Micronucleus Assay on Cryopreserved Whole Blood

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