differential nuclear staining assay: an assay to determine mitocan cytotoxicity by propidium iodide and hoechst double staining

Differential Nuclear Staining Assay: An Assay to Determine Mitocan Cytotoxicity by Propidium Iodide and Hoechst Double Staining

Begin by preparing solutions of the compounds of interest at the desired concentration. Always make sure to mix compounds by vortexing thoroughly.
To measure cytotoxicity of a single compound, prepare compounds at 2X final concentration. If measuring cytotoxicity of compound combinations, prepare them at a 4X final concentration.
Prepare solvent-only controls by mixing the same amount of solvent with the appropriate medium. Collect cells into a 15-milliliter conical tube, and aliquot 10 microliters of the cell suspension for trypan blue staining. Add 10 microliters of 0.4% trypan blue to the aliquot, and use a hemocytometer or cell counter to count viable and non-viable cells.
Pellet cells in the 15-milliliter tube at 200 x g for 5 minutes, then, aspirate or decant the supernatant. Resuspend the cell pellet in assay-appropriate media at a cell density of 3x105 cells per milliliter. Use a multi-channel pipette to seed 50 microliters of cell suspension into each well of a 96-well plate.
For single compound assays, add 50 microliters of 2X compound solution into each well. For solvent-control wells, add 50 microliters of test media containing the solvent at 2X concentration. For combination assays, add 25 microliters of each of the 4X compounds into each well. For single compound-control wells, add 25 microliters each of 4X compound solution and test medium. Make sure that the final concentration of DMSO does not exceed 0.5%.
To ensure the reproducibility, add drugs with the pipette tip touching the side of wells, being careful to add the drugs to different locations. This step should be performed very quickly, preferably taking no longer than 30 minutes per plate.
Gently tap the plate to mix the contents of the wells, and incubate at 37 degrees Celsius and 5% carbon dioxide.
When ready to stain the cells with Hoechst 33342 and propidium iodide, prepare fresh 10X staining solution, and add 10 microliters to each well. Gently tap the plate, and incubate it at 37 degrees Celsius for 15 minutes.
Centrifuge the plate at 200 x g for 4 minutes to bring all the cells to the bottom of the plate. Then, carefully wipe the bottom of the plate with a damp laboratory wipe to remove any debris that may interfere with imaging. Image the plate within 15 minutes after centrifugation.

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1. Cytotoxicity Assay: Setup
<ol>
	<li>Prepare solutions of compounds of interest at desired concentrations in the appropriate media (serum-free or 1, 2.5, or 5% FBS RPMI-1640).
	<ol>
		<li>To measure the cytotoxicity of a single compound (e.g., to determine effective doses), prepare compounds at 2x final concentration.</li>
		<li>To measure the cytotoxicity of compound combinations, prepare compounds at 4x final concentration.</li>
		<li>Prepare solvent-only controls by mixing the same amount of solvent with the appropriate medium. For example, if testing compounds dissolved in DMSO and methanol, make solvent-only control for each solvent.</li>
	</ol>
	</li>
	<li>Collect cells from the culture dish or flask into a 15 mL conical tube.</li>
	<li>Transfer 10 &#181;L of cell suspension into a microcentrifuge tube and stain with 10 &#181;L 0.4% trypan blue. Use a hemocytometer to count viable and non-viable cells for each cell source.</li>
	<li>Pellet cells at 200 g for 5 min. Aspirate or decant supernatant.</li>
	<li>Resuspend cell pellet in assay-appropriate media (serum-free or 1, 2.5, 5% FBS RPMI-1640) at a cell density of 3*105 cells/mL. 
	NOTE 1: Cell density of 3*105 cells/mL provides a seeding density of 15,000 cells/well. Seeding density is an important parameter and ideally should be pre-defined prior to the experiment. Seeding density should take into account 1) cell size &#8211; usually larger cells are seeded at a lower density; 2) treatment duration &#8211; cells are typically seeded at a lower density for experiments that will last longer, and 3) cell division rate &#8211; cells with a higher rate of division are seeded at a lower density. Specific examples of optimized seeding densities: K562 cells, larger, 24 h duration &#8211; 10,000 cells/well; MOLM-13 cells, moderate size, 24 h treatment &#8211; 15,000/well; MOLM-13 cells, 48 h treatment &#8211; 8,000/well; small healthy peripheral blood mononuclear cells (PBMCs), 24 h treatment &#8211; 50,000/well; primary AML cells, 24 h treatment &#8211; 15,000-20,000/well. 
	NOTE 2: The presence of FBS in media may affect the activity of the compounds. Reducing the concentration of FBS may make assay results simpler to interpret, but it also reduces the physiological accuracy.</li>
	<li>Seed 50 &#181;L of cell suspension from step 1.5 into each well of a 96-well plate using a multichannel pipette.</li>
	<li>Add compounds as follows:
	<ol>
		<li>For single compound assays, add 50 &#181;L of 2x compound solution into each well. For solvent-control wells, add 50 &#181;L of test media containing the solvent at the 2x concentration.</li>
		<li>For combination assays, add 25 &#181;L of each of the compounds (4x solutions) into each well. For single compound-control wells, add 25 &#181;L of 4x compound solution and 25 &#181;L of test medium. For solvent-control wells, add 50 &#181;L of test medium or test medium containing the solvent. 
		NOTE 1: The final concentration of DMSO should not exceed 0.5%. 
		NOTE 2: It is recommended to add the media containing the compounds with the pipette touching the wall of each well due to low volume.</li>
	</ol>
	</li>
	<li>Gently tap the plate to ensure the mixing of the contents of the wells.</li>
	<li>Incubate plates at 37 &#176;C in a humidified 5% CO2 atmosphere for an appropriate time, e.g., 24 h.</li>
</ol>
2. Cytotoxicity Assay: Staining with Hoechst 33342 and Propidium Iodide
<ol>
	<li>Prepare 10x staining solution. This solution needs to be prepared fresh before each experiment, it cannot be stored. Final dye concentrations should be determined prior to the experiment.
	<ol>
		<li>For leukemia cell lines and primary leukemia cells, 1 mL of 10x staining buffer contains 10 &#181;L of 20 mM Hoechst 33342 and 50 &#181;L of 1 mg/mL propidium iodide in sterile PBS (final concentrations: Hoechst 33342 20 &#181;M, PI 5 &#181;g/mL).</li>
		<li>For healthy PBMCs, 1 mL of 10x staining buffer contains 10 &#181;L of 20 mM Hoechst 33342 and 10 &#181;L of 1 mg/mL propidium iodide in sterile PBS (final concentrations: Hoechst 33342 20 &#181;M, PI 1 &#181;g/mL). 
		NOTE: The final concentration of propidium iodide has to be determined prior to the experiments. Cells should be tested using a range of PI concentrations (1, 2.5, 5 &#181;g/mL), and then Hoechst/PI-calculated viability should be compared with viability measured via trypan blue. The PI concentrations listed above were chosen based on target cell viability in media-control wells (above ~90% for leukemia cell lines, above ~70% for healthy PBMCs). 
		CAUTION: Hoechst 33342 and propidium iodide are potential carcinogens. Wear appropriate personal protective equipment when handling them.</li>
	</ol>
	</li>
	<li>After incubation, use a multichannel pipette to add 10 &#181;L of 10x staining buffer to each well. 
	NOTE: To prevent cross-contamination, make sure that the pipette tips do not touch the media.</li>
	<li>Gently tap the plate to mix and clear bubbles. Stain at 37 &#176;C for 15 min.</li>
	<li>Centrifuge the plate at 200 g for 4 min to bring all of the cells to the bottom of the plate. Carefully wipe the bottom of the plate with a damp kimwipe to remove fibers and/or debris that will interfere with imaging. 
	NOTE 1: Centrifugation of the plate ensures the highest chances for all of the cells to be captured in the image. Often dead cells will detach and float, providing misleading values of cytotoxicity. Centrifugation mitigates this effect. 
	NOTE 2: The plate should be imaged as quickly as possible after centrifugation, ideally, within 15 min. Centrifugation can reduce the selectivity of PI staining and may allow cells to slowly accrue propidium iodide. The improvement in accuracy gained by visualizing the dead cells outweighs the slight increase in PI staining</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Deoxy-D-glucose/2-DG</td>
			<td>Chem-Impex</td>
			<td>50-519-067</td>
			<td></td>
		</tr>
		<tr>
			<td>3-bromo-pyruvate</td>
			<td>Alfa Aesar</td>
			<td>1113-59-3</td>
			<td></td>
		</tr>
		<tr>
			<td>96-Well plates</td>
			<td>Greiner Bio-One</td>
			<td>655090</td>
			<td>Black or clear flat-bottomed 96-well plates</td>
		</tr>
		<tr>
			<td>Countess II automated cell counter</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cytation 5 Cell Imaging Multi-Mode Reader</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Thermo Fisher</td>
			<td>62249</td>
			<td>20 mM solution; final concentration 1:1,000</td>
		</tr>
		<tr>
			<td>HyClone fetal bovine serum</td>
			<td>GE Healthcare</td>
			<td>#25-514</td>
			<td></td>
		</tr>
		<tr>
			<td>m-chlorophenylhydrazone/CCCP</td>
			<td>Sigma Aldrich</td>
			<td>C2759</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS tablets</td>
			<td>Thermo Fisher</td>
			<td>BP2944100</td>
			<td>1 tablet + 200 mL of sterile water = 1x PBS solution</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin-Glutamine (100X)</td>
			<td>Gibco</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Thermo Fisher</td>
			<td>50-596-072</td>
			<td>Dry powder; stock 1 mg/mL in PBS; final concentration 5 &#181;g/mL (leukemia cells), 1 &#181;g/mL (normal PBMCs)</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Ark Pharm</td>
			<td>AK115691</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 Medium With L-glutamine and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture</td>
			<td>Sigma Aldrich</td>
			<td>R8758-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue stain (0.4%)</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>K562</td>
			<td>ATCC</td>
			<td>CCL-243</td>
			<td>CML cell line</td>
		</tr>
		<tr>
			<td>MOLM-13</td>
			<td>ATCC</td>
			<td></td>
			<td>AML cell line</td>
		</tr>
		<tr>
			<td>MOLT-4</td>
			<td>ATCC</td>
			<td>CRL-1582</td>
			<td>ALL cell line</td>
		</tr>
		<tr>
			<td>OCI-AML2</td>
			<td>ATCC</td>
			<td></td>
			<td>AML cell line</td>
		</tr>
	</tbody>
</table>

Source: Pei, J. et al., <a href="https://www.jove.com/v/61295">An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity.</a> J. Vis. Exp. (2020)
This video demonstrates a nuclear staining method to detect the cytotoxic effect of mitocan on drug-sensitive cancer cells and healthy variants. The double staining with DNA-binding dyes such as propidium iodide and Hoechst dye provides a clear distinction between the dead and live cells, thus helping to assess the cytotoxicity of the drug.

Source: Pei, J. et al., An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity. J. Vis. Exp. (2020)
This ...

Mitocans are a class of drugs that selectively affect the functioning of mitochondria in cancer cells and induce apoptosis.
To determine mitocan cytotoxicity, seed a cell suspension containing semi-adherent cancer cells onto a multi-well plate. Treat the plate with the desired mitocan solution and incubate.
Mitocan enters the cancer cell to disrupt its mitochondrial activity, eventually leading to cell death in mitocan-sensitive cancer cells. Treat the cells with a staining solution containing a mixture of Hoechst dye and propidium iodide.
Hoechst, a membrane-permeable fluorescent dye, penetrates all the cells and binds to the AT-rich region of their DNA, imparting a blue fluorescence to the nuclei of both live and dead cells.
In contrast, propidium iodide, a membrane-impermeable fluorescent dye, exclusively enters the dead cells through the damaged portions of their membranes. The dye intercalates between the DNA bases, imparting an additional red fluorescence to the dead cells. The presence of both dyes in the nuclei of these cells make the dead cells appear purple.
Centrifuge the plate at a low speed to settle the cells for enhanced accuracy during the imaging. Image under a fluorescence microscope, and observe two distinct cell populations. Count the number of both cell types.
The percentage of purple cells representing the dead population compared to the total population indicates the cytotoxic potential of the test mitocan.

146 조회수. Differential Nuclear Staining Assay: Propidium Iodide 및 Hoechst Double Staining에 의한 미토칸 세포 독성을 측정하기 위한 분석

Differential Nuclear Staining Assay: An Assay to Determine Mitocan Cytotoxicity by Propidium Iodide and Hoechst Double Staining

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