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Cat. No.: CLIS-50 (for 100T)

Cat. No.: CLIS-200 (for 200T)

Description

Cell Line Identification Kit by STR (Human, 16 Loci)—also known as the Cell Line Authentication STR Profiling Kit or Cell STR Testing Kit, and referred to in short as the Human Cell Line STR Identification Kit or Cell STR Identification Kit—is a reagent kit designed for rapid, accurate, and highly sensitive STR genotyping of human-derived cell lines. It utilizes multiplex fluorescent PCR and capillary electrophoresis across up to 16 STR loci. The kit can also be applied to forensic analysis or kinship testing of human samples.

The detection principles and methodology of this product are essentially consistent with Thermo Fisher’s Applied Biosystems CLA IdentiFiler™ Plus PCR Amplification Kit (A44660) and Promega’s PowerPlex® 16 HS System (DC2100/DC2101).

Cell line authentication refers to the identification of model cell lines widely used in scientific research and drug development. The use of misidentified or cross-contaminated cell lines can lead to erroneous conclusions, irreproducible results, and serious clinical failures in cell-based therapies. In recent years, organizations such as NIH, ATCC, Nature, and Science have repeatedly called on researchers to authenticate cell lines to prevent such issues.

STR genotyping has been adopted as the gold standard for cell line authentication by authoritative bodies such as ICLAC and ATCC. STR (Short Tandem Repeat) loci consist of short tandem repeats of 2–7 base pairs, which are widely distributed throughout the human genome. These highly polymorphic markers are considered the DNA fingerprint of cells. Alleles at STR loci can be distinguished by the number of repeat units amplified via PCR. The resulting STR profiles are compared against cell line STR databases to identify the sample’s origin or detect possible cross-contamination.

Cross-contamination between cell lines or misidentification during cultivation is a persistent issue. Studies estimate that the frequency of cell line cross-contamination ranges from 16% to 35%. Therefore, cell line authentication is recommended under the following circumstances:

1. When introducing a new cell line into the lab

2. After prolonged cultivation (e.g., >10 passages)

3. Before registering a new cell bank or newly constructed cell line

4. When phenotypic changes occur or experimental results deviate significantly from expectations

5. Prior to clinical trials involving cell use

6. At the start or conclusion of cell-related projects

7. Before using long-frozen or poorly labeled cell lines in experiments

The polymorphism or informativeness of STR markers reflects differences in repeat unit numbers between alleles and loci from different sources, as illustrated in Figure 1. In diploid cell lines, each chromosomal locus typically contains two allele copies—one from the maternal chromosome (M) and one from the paternal chromosome (P). If both alleles have the same number of repeat units, the PCR product will be a single fragment of uniform size, appearing as a single peak in capillary electrophoresis. If the alleles differ in repeat unit count, two distinct fragment sizes will be produced, resulting in two peaks.

Figure 1. Schematic Diagram of STR Polymorphism Analysis

STR results can be compared against databases from ATCC, DSMZ, and the China National Cell Resource Sharing Platform. Currently, the ATCC and DSMZ databases typically use STR data from 9 loci—including 8 core STR loci and the Amelogenin sex-determining locus—to calculate the match percentage between the test sample and reference cell lines. A match rate of 80% or higher is generally considered sufficient to confirm the identity of the cell line.

Additional STR loci beyond these core sites enhance the accuracy of cell line identification and improve resolution between closely related cell lines. The greater the number of loci analyzed, the higher the precision and discriminatory power of the authentication process.

For reference, the results of multiplex fluorescent PCR and capillary electrophoresis performed using this kit on the PCR control template—HT1080 cells (human fibrosarcoma cell line) genomic DNA—are shown in Figure 2. The corresponding comparison results from the DSMZ database are presented in Figure 3.

Figure 2: STR Genotyping Results of HT1080 Cells Using the Cell Line STR Identification Kit (Human, 16 Loci)

This figure illustrates the STR genotyping performance of the Cell Line STR Identification Kit (Human, 16 Loci) when applied to HT1080 cells (human fibrosarcoma cell line). A total of 200 ng of HT1080 genomic DNA was used as the template for multiplex fluorescent PCR amplification. The resulting products were analyzed using an ABI 3730xl DNA Analyzer equipped with a 50 cm capillary.

• X-axis: Number of base pairs (bp) of the PCR products and internal size standard GeneScan™ 500 LIZ®

• Y-axis: Relative fluorescence intensity

Peak color coding and corresponding fluorophores:

• 🔵 Blue peaks (FAM-labeled loci): D3S1358, TH01, D21S11, D18S51, Penta E

• 🟢 Green peaks (HEX-labeled loci): D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D

• ⚫ Black peaks (TAMRA-labeled loci): Amelogenin (AMEL/AME/AM), vWA, D8S1179, TPOX, FGA

• 🟠 Orange peaks: GeneScan™ 500 LIZ® internal size standard fragments ranging from 100 bp to 430 bp

Allele calls for each locus can be found in Section 4, “Result Interpretation and Analysis,” of the kit’s user manual. Please note that actual detection results may vary depending on sample quality, instrumentation, and other experimental conditions. The data shown in this figure is for reference only.

Figure 3. Cell Line Authentication Results of HT1080 Cells Using This STR Kit

This figure presents the cell line authentication results for HT1080 cells (human fibrosarcoma cell line) using the Cell Line STR Identification Kit. The STR profile data obtained from the analysis was submitted to the DSMZ database for comparison. The results showed a 96% match with the HT1080 reference profile, confirming the identity of the cell line. The presence of multiple alleles at the D13S317 locus may indicate cellular variation. The ATCC catalog number for HT1080 cells is CCL-121. Please note that actual detection results may vary depending on sample quality, instrumentation, and other experimental conditions. The data shown in this figure is for reference only.

This kit is based on multiplex fluorescent PCR and capillary electrophoresis, enabling simultaneous amplification and three-color fluorescence detection of 16 loci in human-derived cells (15 STR loci and 1 sex-determining locus). The loci included in the assay are:

• FAM-labeled loci: D3S1358, TH01, D21S11, D18S51, Penta E

• HEX-labeled loci: D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D

• TAMRA-labeled loci: Amelogenin (sex-determining locus), vWA, D8S1179, TPOX, FGA

A total of 16 loci are analyzed, with Amelogenin serving as the sex marker. Detailed information is provided in the table below.

This kit provides all necessary reagents for the amplification of the 16 loci listed above. After PCR amplification, users can perform capillary electrophoresis to generate allele profiles of the test samples for final result interpretation.

The kit also includes a PCR control template (genomic DNA from HT1080 cells), which serves as a quality control to verify proper kit performance and can be used as a reference during result analysis.

According to the instructions, when the reaction volume is set to 10 µl, the small and medium package formats of this kit support approximately 50 and 200 reactions, respectively.

Components

• Multiplex STR PCR Master Mix (4X)
• Human 16 Loci Primer Mix (4X)
• Control Template (400ng/µl)
• Ultrapure Water

Storage

Store at –20ºC. Valid for one year. The Human 16 Loci Primer Mix (4X) must be protected from light during storage.

Precautions

• This kit provides reagents required only for multiplex fluorescent PCR. For capillary electrophoresis and data analysis, please contact our technical support at tech@sbsbio.com.

• We also offer a high-efficiency PCR premix specifically designed for multiplex STR genotyping: Multiplex STR PCR Master Mix (4X).

• It is recommended to use filter tips when preparing the PCR reaction system to minimize contamination.

• The Human 16 Loci Primer Mix (4X) contains fluorescent dyes. Avoid exposure to strong light during storage and PCR setup to prevent fluorescence quenching.

• The kit has been tested to withstand up to 10 freeze-thaw cycles without significant performance loss. However, repeated freeze-thawing should still be avoided to maintain optimal reagent quality.

• After multiplex fluorescent PCR, samples must be analyzed using a capillary electrophoresis instrument. Note that allele sizes may vary depending on the polymerase and instrument used.

• Due to the kit’s high sensitivity, it is strongly recommended to separate the following areas into three distinct rooms to prevent contamination:

  1. Sample preparation

  2. PCR reaction setup

  3. Post-PCR product handling and electrophoresis

  The area used for opening PCR tubes and performing capillary electrophoresis should be physically separated from the sample preparation and PCR setup areas.

• For assistance with capillary electrophoresis, data analysis, or direct STR-based cell line authentication, please contact our technical support at tech@sbsbio.com.

• This product is intended exclusively for scientific research by trained professionals. It must not be used for clinical diagnosis or treatment, not be applied to food or pharmaceuticals, and must not be stored in residential environments.

• For your safety and health, please wear a lab coat and disposable gloves when handling this product.

Only for research and not intended for treatment of humans or animals

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