Name: Colony Formation Assay Detecting the Proliferative Capacity of LncRNA-knockdown Osteosarcoma Cells (Video) | JoVE Journal
Uploaded: 2026-01-16T13:45:00.000+00:00
Duration: 6 min 13 s
Description: Learn colony formation assays to assess lncRNA-knockdown effects on osteosarcoma cell proliferation using 143B cells. This protocol enables reliable quantification of long-term cancer cell growth.

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5 minutes

This work was supported by grants from the National Nature Science Foundation (82174408, 82374477, 82474535, and&#160;82205145).

The reagents and the equipment used are listed in the Table of Materials.
1. Plasmid transfection
<ol>
	<li>Seed 5 &#215; 105 143B cells per 10 cm culture dish (total two dishes), culture the cells in complete medium (DMEM + 10% FBS) at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Prepare transfection reagents 24 h later, including plasmids (shNC and shSNHG6), PEI, and Opti-MEM. 
	NOTE: Vortex and ensure all are equilibrated to room temperature before use.</li>
	<li>Take out four 1.5 mL microcentrifuge tubes, label the plasmids (shNC and shSNHG6) and PEI (two tubes) using a marker pen.</li>
	<li>Add 500 &#181;L of Opti-MEM to each tube.</li>
	<li>Add 10 &#181;g of plasmids to the plasmid tube (shNC or shSNHG6) and add 30 &#181;L of PEI (1&#181;g/&#181;L) to each PEI tube.</li>
	<li>Mix gently up and down, incubate at room temperature for 5 min.</li>
	<li>Combine the plasmid tube and PEI tube, mix gently, and incubate the mixture at room temperature for 20 min.</li>
	<li>Take out 143B cells from the incubator and aspirate the culture medium gently. Add 7 mL of serum-free medium to each dish. Label the dishes to indicate the plasmids to be added (143B shNC or 143B shSNHG6).</li>
	<li>Add the pre-prepared transfection mixture dropwise to the corresponding culture dish. 
	NOTE: Add the mixture slowly, using a pipette in a circular motion, and evenly dispense it from the outer edge toward the center of the dish. Incubate at 37 &#176;C in an incubator with 5% CO2.</li>
	<li>Discard the supernatant 8-12 h later, and add 10 mL of complete DMEM medium to each 10 cm culture dish. Incubate the cells at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Observe under fluorescence microscope at 72 h after the supernatant is discarded and replaced with complete DMEM medium.</li>
</ol>
2. Colony formation assay
<ol>
	<li>Remove the medium from the 10 cm culture dish (shNC and shSNHG6), add 1.5 mL trypsin-EDTA to digest the cells, gently pipette to detach them, and then add 5mL of complete medium to terminate the digestion.</li>
	<li>Transfer the cell suspension into a 15 mL microcentrifuge tube.</li>
	<li>Take out 20 &#181;L of cell suspension and count cell numbers. 
	NOTE: The cell count must be performed multiple times to ensure that the cell numbers in the shNC group and the shSNHG6 group are consistent. Here, we collect 3 &#215; 105 cells for RT-PCR verification of SNHG6 knockdown efficiency.</li>
	<li>Seed 2 &#215; 103 cells per well in a 6-well plate. Add complete DMEM medium to a final volume of 2 mL and culture the cells at 37 &#176;C in a 5% CO2 incubator. 
	NOTE: Replace the medium with fresh complete DMEM medium every other day.</li>
	<li>Monitor colony formation daily. 
	NOTE: Terminate the culture when most colonies in the control (shNC) group contain &#8805;50 cells, or after 10-14 days of incubation, whichever comes first.</li>
	<li>Take out cells from the incubator and discard the supernatant.</li>
	<li>Wash cells twice with 1 mL of PBS per well, and discard the PBS after each wash.</li>
	<li>Fix cells with 1 mL of 10% formalin solution for 15 min, then discard the 10% formalin solution.</li>
	<li>Wash cells twice with 1 mL of PBS per well, and discard the PBS after each wash.</li>
	<li>Stain cells with 1 mL of 0.5% crystal violet staining solution for 10-30 min, then discard the staining solution.</li>
	<li>Wash cells gently with slowly running tap water.</li>
	<li>Air-dry cells at room temperature.</li>
	<li>Capture images using a camera.</li>
</ol>

<ol><li>Chen, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IAV+inhibits+the+host+antiviral+defense+mediated+by+LncRNA-LRIR+to+enhance+viral+replication%5BTitle%5D)+AND+%22Int+J+Biol+Macromol%22%5BJournal%5D)">IAV inhibits the host antiviral defense mediated by LncRNA-LRIR to enhance viral replication.</a> Int J Biol Macromol. 322 (Pt 1), 146665(2025).</li>
<li>Zhu, J., Mo, Y. Y., Peng, W. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA+binding+protein-mediated+competing+endogenous+RNA+mechanism+in+cancer%5BTitle%5D)+AND+%22Gene%22%5BJournal%5D)">RNA binding protein-mediated competing endogenous RNA mechanism in cancer.</a> Gene. 963, 149606(2025).</li>
<li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((KIAA1429-mediated+ZFPM2-AS1+m%286%29A+modification+promotes+the+proliferation%2C+migration+and+invasion+of+osteosarcoma+cells%5BTitle%5D)+AND+%22Oncol+Lett%22%5BJournal%5D)">KIAA1429-mediated ZFPM2-AS1 m(6)A modification promotes the proliferation, migration and invasion of osteosarcoma cells.</a> Oncol Lett. 30 (4), 453(2025).</li>
<li>Oh, M., Kadam, R. N., Charania, Z. S., Somarowthu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((LncRNA+SChLAP1+promotes+cancer+cell+proliferation+and+invasion+via+its+distinct+structural+domains+and+conserved+regions%5BTitle%5D)+AND+%22J+Mol+Biol%22%5BJournal%5D)">LncRNA SChLAP1 promotes cancer cell proliferation and invasion via its distinct structural domains and conserved regions.</a> J Mol Biol. 437 (19), 169350(2025).</li>
<li>Wang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TBX5-AS1+induces+ER+stress+and+suppresses+lung+cancer+growth+and+tumor+stemness+via+the+miR-494-3p%2FATF6+axis%5BTitle%5D)+AND+%22Cell+Stress+Chaperones%22%5BJournal%5D)">TBX5-AS1 induces ER stress and suppresses lung cancer growth and tumor stemness via the miR-494-3p/ATF6 axis.</a> Cell Stress Chaperones. 30 (6), 100113(2025).</li>
<li>Xu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((lncRNA+POU3F3+promotes+osteosarcoma+progression+through+GPX4-modulated+ferroptosis+by+interaction+with+IGF2BP2+to+facilitate+NRF2+mRNA+stability%5BTitle%5D)+AND+%22Genes+Dis%22%5BJournal%5D)">lncRNA POU3F3 promotes osteosarcoma progression through GPX4-modulated ferroptosis by interaction with IGF2BP2 to facilitate NRF2 mRNA stability.</a> Genes Dis. 12 (5), 101439(2025).</li>
<li>Li, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((LncRNA+HOXA-AS3+promotes+cell+proliferation+and+invasion+via+targeting+miR-218-5p%2FFOXP1+axis+in+osteosarcoma%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">LncRNA HOXA-AS3 promotes cell proliferation and invasion via targeting miR-218-5p/FOXP1 axis in osteosarcoma.</a> Sci Rep. 14 (1), 16581(2024).</li>
<li>Ghasemi, M., Turnbull, T., Sebastian, S., Kempson, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+MTT+assay%3A+Utility%2C+Limitations%2C+pitfalls%2C+and+interpretation+in+bulk+and+single-cell+analysis%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">The MTT assay: Utility, Limitations, pitfalls, and interpretation in bulk and single-cell analysis.</a> Int J Mol Sci. 22 (23), (2021).</li>
<li>Harris, L., Zalucki, O., Piper, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BrdU%2FEdU+dual+labeling+to+determine+the+cell-cycle+dynamics+of+defined+cellular+subpopulations%5BTitle%5D)+AND+%22J+Mol+Histol%22%5BJournal%5D)">BrdU/EdU dual labeling to determine the cell-cycle dynamics of defined cellular subpopulations.</a> J Mol Histol. 49 (3), 229-234 (2018).</li>
<li>Romar, G. A., Kupper, T. S., Divito, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Research+techniques+made+simple%3A+Techniques+to+assess+cell+proliferation%5BTitle%5D)+AND+%22J+Invest+Dermatol%22%5BJournal%5D)">Research techniques made simple: Techniques to assess cell proliferation.</a> J Invest Dermatol. 136 (1), e1-e7 (2016).</li>
<li>Stefanowicz-Hajduk, J., Ochocka, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-time+cell+analysis+system+in+cytotoxicity+applications%3A+Usefulness+and+comparison+with+tetrazolium+salt+assays%5BTitle%5D)+AND+%22Toxicol+Rep%22%5BJournal%5D)">Real-time cell analysis system in cytotoxicity applications: Usefulness and comparison with tetrazolium salt assays.</a> Toxicol Rep. 7, 335-344 (2020).</li>
<li>Franken, N. A., Rodermond, H. M., Stap, J., Haveman, J., van Bree, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clonogenic+assay+of+cells+in+vitro%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Clonogenic assay of cells in vitro.</a> Nat Protoc. 1 (5), 2315-2319 (2006).</li>
<li>Brix, N., Samaga, D., Belka, C., Zitzelsberger, H., Lauber, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+clonogenic+growth+in+vitro%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Analysis of clonogenic growth in vitro.</a> Nat Protoc. 16 (11), 4963-4991 (2021).</li>
<li>Rajendran, V., Jain, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+tumorigenic+assay%3A+Colony+forming+assay+for+cancer+stem+cells%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">In vitro tumorigenic assay: Colony forming assay for cancer stem cells.</a> Methods Mol Biol. 1692, 89-95 (2018).</li>
<li>Guzman, C., Bagga, M., Kaur, A., Westermarck, J., Abankwa, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ColonyArea%3A+An+ImageJ+plugin+to+automatically+quantify+colony+formation+in+clonogenic+assays%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">ColonyArea: An ImageJ plugin to automatically quantify colony formation in clonogenic assays.</a> PLoS One. 9 (3), e92444(2014).</li>
</ol>

The authors have nothing to disclose.

Therapeutically targeting cancer-associated lncRNAs, in addition to leveraging their diagnostic utility as biomarkers, represents an emerging approach in cancer management. Defining the roles of lncRNAs in osteosarcoma pathogenesis is crucial for advancing its diagnosis, therapy, and prognosis.
Critical steps must be followed to ensure the success of this assay. First, establishing a single-cell suspension is an absolute prerequisite for reliable clonal assays, as the presence of cell aggregat...

Long non-coding RNAs (lncRNAs), defined as transcripts longer than 200 nucleotides without protein-coding potential, have emerged as important regulators of diverse biological processes in recent years1,2. They play critical roles in the initiation and development of various cancers, including osteosarcoma, prostate cancer, and lung cancer3,4,5. In osteosarcoma, accumulating evidence suggests that aberrant long non-coding RNA (lncRNA) expression has a profound influence on cellular proliferation. For instance, silencing lncRNA POU3F3 significantly suppressed the proliferation and promoted the apoptosis of osteosarcoma cells (MG63 and U2OS)6. Similarly, knockdown of lncRNA HOXA-AS3 reduced colony formation and inhibited growth in U2OS and SW1353 cells7. Given that uncontrolled proliferation is a hallmark of cancer biology and a major determinant of therapeutic strategies, elucidating lncRNA-regulated cell proliferation in tumors, particularly in osteosarcoma, remains of great importance and necessity.
Although several approaches exist to evaluate cell proliferation, each carries inherent limitations. Colorimetric or metabolic assays, such as CCK-8 and MTT, are convenient, rapid, and high-throughput. However, they primarily provide short-term readouts and are easily confounded by metabolic fluctuations, and have additional risks of optical or photochemical interference from the reagents used8. DNA synthesis-based methods, including BrdU and EdU incorporation, offer greater specificity while limiting to snapshot analyses of S-phase entry without reflecting long-term proliferative outcomes9,10. Similarly, immunostaining for proliferation markers, such as Ki-67, only indicates instantaneous cell cycle activity and fails to predict clonogenic capacity10. Real-time monitoring systems, such as RTCA, enable continuous growth curve analysis with high reproducibility, yet their high cost and restriction to short- or intermediate-term assessment reduce their practicality11. By contrast, the clonogenic assay uniquely captures the capacity of a single cell to undergo unlimited proliferation and form visible colonies, thereby directly reflecting its ultimate fate. Despite being labor-intensive and time-consuming, the clonogenic assay remains the gold standard for assessing long-term proliferative potential, offering insights that are unattainable by short-term or surrogate assays12.
Here, this article includes a detailed detection protocol for lncRNA-knockdown and colony formation assays, with an example of SNHG6-knockdown in 143B osteosarcoma cells. This approach is not only applicable to osteosarcoma research but also serves as a feasible tool for investigating lncRNA-mediated proliferation in other tumors.

<table><tbody><tr><td>1.5 mL MICROTUBES</td><td>Merck</td><td>AXYMCT150C</td><td></td></tr><tr><td>10% Neutral Buffered Formalin</td><td>VICTREX</td><td>5205000</td><td></td></tr><tr><td>100 - 1000 &amp;mu;L Extended Graduated Blue Tips</td><td>Shanghai BioScience Co., Ltd</td><td>BS10401ML</td><td></td></tr><tr><td>100mm Cell Culture Dishes laboratory</td><td>Wuxi NEST Biotechnology Co.,Ltd</td><td>704001</td><td></td></tr><tr><td>143B</td><td>ATCC</td><td>ATCC CRL-8303TM</td><td></td></tr><tr><td>15 mL Centrifuge Tube</td><td>Shanghai BioScience Co., Ltd</td><td>BL2002150</td><td></td></tr><tr><td>6 Well Cell Culture Plate</td><td>Wuxi NEST Biotechnology Co.,Ltd</td><td>703001</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo Fisher Technology (China)Co., Ltd.</td><td>300583057</td><td></td></tr><tr><td>Crystal Violet</td><td>TCI Shanghai</td><td>C0428</td><td></td></tr><tr><td>DMEM medium</td><td>WISENT CORPORATION</td><td>319-005-CL</td><td></td></tr><tr><td>Eppendorf Research plus</td><td>Eppendorf</td><td>3123000233</td><td></td></tr><tr><td>FBS - Superior quality</td><td>WISENT CORPORATION</td><td>086-150</td><td></td></tr><tr><td>Mark pen</td><td>Zebra Trading (Shenzhen) Co.,Ltd.&amp;nbsp;</td><td>YYST5</td><td></td></tr><tr><td>Opti-MEM</td><td>Gibco</td><td>31985070</td><td></td></tr><tr><td>PASTEUR PIPETTE</td><td>Labgic</td><td>BS-XG-O3L</td><td></td></tr><tr><td>PBS buffer (dry powder)</td><td>Labgic</td><td>BL601A</td><td></td></tr><tr><td>PEI</td><td>Polysciences</td><td>24765</td><td></td></tr><tr><td>Pipette tips</td><td>Merck</td><td>AXYT200Y</td><td></td></tr><tr><td>TrypLE Express Enzyme</td><td>Gibco</td><td>12604021</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Gibco</td><td>R001100</td><td></td></tr></tbody></table>

colony formation assay detecting the proliferative capacity of lncrna-knockdown osteosarcoma cells

Colony Formation Assay Detecting the Proliferative Capacity of LncRNA-knockdown Osteosarcoma Cells

Aberrant long non-coding RNAs (lncRNAs) expression profoundly influences cellular proliferation of osteosarcoma cells, suggesting that they may serve as potential therapeutic targets. However, functional studies of lncRNAs remain largely theoretical and limited to a few validations. Colony formation assays, which specifically reflect the ultimate proliferative fate of single cells, are regarded as the gold standard for evaluating long-term proliferative potential. Here, this protocol established a reliable experimental protocol using colony formation assays in lncRNA-knockdown cells to evaluate the effect of lncRNA on osteosarcoma cell proliferative capacity. This experimental protocol provides a stable, cost-effective, and efficient assay to assess the regulatory effects of lncRNA on osteosarcoma cell proliferation. This approach is not only suitable for research on osteosarcoma cells but also serves as a feasible tool for lncRNA-regulated cell proliferative capacity in other tumors. This study included a detailed detection protocol of lncRNA-knockdown and colony formation assays, with an example of the small nucleolar RNA host gene 6 (SNHG6) knockdown in osteosarcoma cells (143B). Experimental results confirmed efficient suppression of SNHG6 expression, accompanied by a marked reduction in both the number and size of colonies. These findings suggest that SNHG6 is essential for sustaining the long-term proliferative potential of osteosarcoma cells (143B).

To evaluate the effect of lncRNA SNHG6 on the proliferative capacity of osteosarcoma cells, we successfully established a plasmid-mediated knockdown system and performed colony formation assays. The experimental workflow included plasmid transfection of 143B cells and colony formation assay (Figure 1).
Transfection efficiency of 143B cells was confirmed by fluorescence microscopy 72 h after transfection (Figure 2). The results showed ...

This experimental protocol provides a stable, cost-effective, and efficient method that combines lncRNA knockdown with colony-forming assays to quantify the effect of lncRNA on osteosarcoma cell proliferation.

Aberrant long non-coding RNAs (lncRNAs) expression profoundly influences cellular proliferation of osteosarcoma cells, suggesting that they may serve as ...

My research focuses on osteosarcoma and aims to investigate how LncRNA-knockdown influences long-term proliferative capacity using colony information assays. To begin, seed 5 times 10 to the power of 5 143B osteosarcoma cells into two 10 centimeter culture dishes. Culture the cells in complete DMEM supplemented with 10%fetal bovine serum at 37 degrees Celsius in a 5%carbon dioxide incubator.Prepare the transfection reagents 24 hours later, including plasmids, polyethylenimine, and OptiMinium Essential Medium. Vortex each reagent and allow them to equilibrate to room temperature before use. Now, label four 1.5 milliliter microcentrifuge tubes for plasmids and polyethylenimine using a marker pen.Pipette 500 microliters of OptiMinium Essential Medium to each tube. Then add 10 micrograms of plasmid to the corresponding plasmid tube and pipette 30 microliters of polyethylenimine into each polyethylenimine tube. Mix each tube gently by pipetting up and down and incubate.Now combine each plasmid tube with its corresponding polyethylenimine tube. Mix gently and incubate the mixture at room temperature for 20 minutes. Gently aspirate the culture medium from the dishes containing the 143B cells.Add seven milliliters of serum-free medium to each dish and label the dishes according to the plasmid to be added. Add the prepared transfection mixture dropwise to the corresponding culture dish. Add the mixture slowly in a circular motion from the outer edge toward the center of the dish.Incubate the cells at 37 degrees Celsius in a 5%carbon dioxide incubator. Discard the supernatant 8 to 12 hours later. Then add 10 milliliters of complete DMEM to the culture dish and incubate.Observe the cells under a fluorescence microscope 72 hours after replacing the medium, Remove the medium from the culture dishes. Add 1.5 milliliters of trypsin-EDTA acid to digest the cells. Cells gently pipette to detach the cells.Then add five milliliters of complete medium to terminate digestion. Transfer the cell suspension into a 15 milliliter microcentrifuge tube. Then pipette 20 microliters of the cell suspension into a hemocytometer to count the cells.Collect 3 times 10 to the power of 5 cells for ribonucleic acid extraction to verify knockdown efficiency. Now seed 2 times 10 to the power of 3 cells per well into a six-well plate. Pipette complete DMEM to a final volume of two milliliters.Culture the cells at 37 degrees Celsius in a 5%carbon dioxide incubator. Monitor colony formation daily. Terminate the culture when most colonies in the control group contain at least 50 cells or after 10 to 14 days of incubation.After discarding the supernatant, wash the cells twice with one milliliter of PBS per well, discarding the solution after each wash. Fix the cells in one milliliter of 10%formalin solution for 15 minutes. Discard the fixative, then wash the cells twice with one milliliter per well of PBS.Stain the cells with one milliliter of 0.5%crystal violet solution for 10 to 30 minutes. Then gently wash the plate with slowly running tap water. Air dry the cells at room temperature.Capture images of the colonies using a camera. Transfection efficiency of 143B cells was confirmed by fluorescence microscopy 72 hours after transfection, indicating efficient plasmid delivery in both conditions. RT-qPCR reaction analysis showed that SNHG6 expression was significantly reduced in 143B cells transfected with shSNHG6 compared with the shNC group.Colony formation assays revealed a significant reduction in the number and size of colonies in the SNHG6 knockdown group compared to the control group. This protocol assesses single-cell long-term proliferation, providing higher biological relevance and more definitive outcomes than short-term metabolic-based proliferation assays. Future studies will expand this protocol to additional LncRNAs and tumor models to systematically explore mechanisms regulating cancer cell proliferative potential.

colony-formation-assay-detecting-proliferative-capacity-lncrna

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Colony Formation Assay Detecting the Proliferative Capacity of LncRNA-knockdown Osteosarcoma Cells

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