Phone:
+1 877 302 8632
Fax:
+1 888 205 9894 (Toll-free)
E-Mail:
orders@antibodies-online.com

CytoSelect™ 96-Well In Vitro Tumor Sensitivity Assay (Soft Agar Colony Formation)

BCA Cell Samples
Catalog No. ABIN2344894
  • Application
    Biochemical Assay (BCA)
    Brand
    CytoSelect™
    Sample Type
    Cell Samples
    Characteristics
    CytoSelect™ 96-well In Vitro Tumor Sensitivity Assay does not involve subjective manual counting of colonies or require a 3-4 week incubation period. Instead cells are incubated only 6-8 days in a proprietary semisolid agar media before being solubilized, transferred and detected by the provided MTT Solution in a microtiter plate reader (see Assay Principle below). The CytoSelect™ 96-well In Vitro Tumor Sensitivity Assay provides a stringent, anchorage- independent model for chemosensitivity testing and potential anticancer drug screening. Each Trial Size kit provides sufficient quantities to perform 24 tests in a 96-well microtiter plate.
    Components
    1. 10X CytoSelect™ Agar Matrix Solution : One 1 mL sterile tube
    2. CytoSelect™ Matrix Diluent : One 1 mL sterile tube
    3. 5X DMEM Solution : One 1.5 mL sterile tube
    4. 1X Matrix Solubilization Buffer : Three 2 mL sterile tubes
    5. Detergent Solution : Two 1.5 mL tubes
    6. MTT Solution : One 0.3 mL tube
    Material not included
    1. Tumor Cells (cancer cell line or cells prepared from solid tumor)
    2. Anticancer Agents (e.g. Taxol, 5-Fluorouracil, anticancer mAb or siRNA)
    3. 37 °C Incubator, 5 % CO2 Atmosphere
    4. Light Microscope
    5. 96-well Microtiter Plate Reader
    6. 37 °C and boiling water baths
  • Application Notes
    Optimal working dilution should be determined by the investigator.
    Comment

    • Screen for effects of anticancer compounds
    • Uses traditional 3D soft agar matrix
    • Fully quantify tumor sensitivity with no manual cell counting
    • Results in 6-8 days, not 3 weeks

    Assay Time
    6 - 8 d
    Reagent Preparation
    • 2X DMEM/20 % FBS Medium: In a sterile tube, dilute the provided 5X DMEM in sterile cell culture grade water to 2X containing 20 % FBS. For example, to prepare a 2.5 mL solution, add 1 mL of 5X DMEM, 0.5 mL of FBS and 1 mL of sterile cell culture grade water. Sterile filter the 2X media to 0.2 μm. Note: You may substitute your own medium in place of the DMEM we provide, but ensure that it is at a 2X concentration.
    • 10X CytoSelect™ Agar Matrix Solution: Heat the Agar Matrix Solution tube to 90-95 °C in a water bath for 30 minutes, or until agar matrix liquefies. Transfer the tube to a 37 °C water bath for 20 minutes and maintain until needed.
    Assay Procedure

    I. Preparation of Base Agar Matrix Layer

    1. Heat the 10X CytoSelect™ Agar Matrix Solution to 90-95 °C in a water bath for 30 minutes, or until agar matrix liquefies. Transfer the tube to a 37 °C water bath for 20 minutes and maintain until needed.
    2. Warm the 2X DMEM/20 % FBS medium (see Preparation of Reagents section) to 37 °C in a water bath. Allow at least 30 minutes for the temperature to equilibrate. 4
    3. According to Table 1 (below), prepare the desired volume of Base Agar Matrix Layer in the following sequence: a. In a sterile tube, add the appropriate volume of 2X DMEM/20 % FBS medium. b. Next, add the corresponding volume of sterile water. Mix well. c. Finally, add the corresponding volume of 10X CytoSelect™ Agar Matrix Solution. Mix well. Note: The 10X CytoSelect™ Agar Matrix Solution is slightly viscous, care should be taken in accurately pipetting the appropriate volume. 2X DMEM/20 % Sterile Water 10X Total Volume of # of Tests in 96- FBS Medium (mL) CytoSelect™ Base Agar Matrix well Plate (50 (mL) Agar Matrix Layer (mL) μL/test) Solution (mL) 0.625 0.5 0.125 1.25 25 Table
      1. Preparation of Base Agar Matrix Layer
      2. After mixing, maintain the Base Agar Matrix Layer at 37 °C to avoid gelation.
      3. Dispense 50 μL of Base Agar Matrix Layer into each well of a 96-well sterile flat-bottom microplate (samples should be assayed in triplicate). Gently tap the plate a few times to ensure the Base Agar Matrix Layer evenly covers the wells. Notes: • Work quickly with the layer to avoid gelation. Also, try to avoid adding air bubbles to the well. • To avoid fast and uneven evaporation that leads to aberrant results, we suggest not using the wells on the plate edge, or filling the edge wells with medium to reduce evaporation.
      4. Transfer the plate to 4 °C for 30 minutes to allow the Base Agar Matrix Layer to solidify.
      5. Prior to adding the Cell Suspension/Agar Matrix Layer (Section II), allow the plate to warm to room temperature for 30 minutes.

      II. Addition of Cell Suspension/Agar Matrix Layer (under sterile conditions)

      1. Heat the 10X CytoSelect™ Agar Matrix Solution to 90-95 °C in a water bath for 30 minutes, or until agar matrix liquefies. Transfer the tube to a 37 °C water bath for 20 minutes and maintain until needed.
      2. Warm the 2X DMEM/20 % FBS medium (see Preparation of Reagents section) and CytoSelect™ Matrix Diluent to 37 °C in a water bath. Allow at least 30 minutes for the temperature to equilibrate.
      3. Harvest and resuspend cells in culture medium at 0.1 - 1 x 106 cells/mL. Keep the cell suspension warm in a 37 °C water bath. 5
      4. According to Table 2 (below), prepare the desired volume of Cell Suspension/Agar Matrix Layer in the following sequence: a. In a sterile tube, add the appropriate volume of 2X DMEM/20 % FBS medium. b. Next, add the corresponding volume of CytoSelect™ Matrix Diluent. Mix well. c. Next, add the corresponding volume of 10X CytoSelect™ Agar Matrix Solution. Mix well. d. Finally, add the corresponding volume of cell suspension. Mix well. Note: The CytoSelect™ Matrix Diluent and 10X CytoSelect™ Agar Matrix Solution are slightly viscous, care should be taken in accurately pipetting the appropriate volumes. 2X CytoSelect™ 10X Cell Total Volume of # of Tests in DMEM/20 % Matrix Diluent CytoSelect™ Suspension Cell Suspension/ 96-well Plate FBS Medium (mL) Agar Matrix (mL) Agar Matrix (75 μL/test) (mL) Solution (mL) Layer (mL) 0.875 0.688 0.188 0.125 1.875 25 Table
      5. Preparation of Cell Suspension/Agar Matrix Layer
      6. After mixing, incubate the Cell Suspension/Agar Matrix Layer at room temperature for 5 minutes.
      7. Immediately dispense 75 μL of Cell Suspension/Agar Matrix Layer into each well of the 96- well plate, already containing the Base Agar Matrix Layer (Section I). Notes: • Work quickly with the layer to avoid gelation, but gently pipette as not to disrupt the base layer integrity. Also, try to avoid adding air bubbles to the well. • Always include negative control wells that contain no cells in the Cell Suspension/Agar Matrix Layer.
      8. Transfer the plate to 4 °C for 20 minutes to allow the Cell Suspension/Agar Matrix Layer to solidify.
      9. Allow the plate to warm to room temperature for 30 minutes.
      10. Add 50 μL of culture medium containing anticancer agents (e.g. Taxol, 5-Fluorouracil, mAb, etc.) to each well.
      11. Incubate the cells for 6-8 days at 37 °C and 5 % CO2. Examine the colony formation under a light microscope.

      III. Quantitation of Anchorage-Independent Growth

      1. Add 125 μL of the 1X Matrix Solubilization Buffer to each well.
      2. Pipette the entire volume of the well 10-12 times to mix thoroughly and solubilize the agar matrix completely. 6
      3. Transfer 100 μL of the mixture to a 96-well microtiter plate.
      4. Add 10 μL of MTT Solution to each well. Pipette each well 7-10 times to ensure a homogeneous mixture.
      5. Incubate the plate for 2-4 hours at 37 °C and 5 % CO2. Note: Under the microscope, a purple precipitate should be visible within the cells.
      6. Add 100 μL of Detergent Solution to each well.
      7. Incubate the plate in the dark for 2-4 hours at room temperature.
      8. Pipette each well 7-10 times to ensure a homogeneous mixture.
      9. Measure the absorbance at 570 nm in a 96-well microtiter plate reader. Cell Dose Curve (optional)
        1. Heat the 10X CytoSelect™ Agar Matrix Solution to 90-95 °C in a water bath for 30 minutes, or until agar matrix liquefies. Transfer the tube to a 37 °C water bath for 20 minutes and maintain until needed.
        2. Warm the 2X DMEM/20 % FBS medium (see Preparation of Reagents section) and CytoSelect™ Matrix Diluent to 37 °C in a water bath. Allow at least 30 minutes for the temperature to equilibrate.
        3. Harvest and resuspend cells in culture medium at 5 - 10 x 106 cells/mL.
        4. Prepare a serial 2-fold dilution in culture medium, including a blank without cells.
        5. Transfer 50 μL of each dilution to a 96-well plate.
        6. According to Table 3 (below), prepare the desired volume of Cell Dose Curve Solution in the following sequence: a. In a sterile tube, add the appropriate volume of 2X DMEM/20 % FBS medium. b. Next, add the corresponding volume of sterile water. Mix well. c. Next, add the corresponding volume of CytoSelect™ Matrix Diluent. Mix well. d. Finally, add the corresponding volume of 10X CytoSelect™ Agar Matrix Solution. Mix well. Note: The CytoSelect™ Matrix Diluent and 10X CytoSelect™ Agar Matrix Solution are slightly viscous, care should be taken in accurately pipetting the appropriate volumes. 2X Sterile Water CytoSelect™ 10X Total Volume of DMEM/20 % (mL) Matrix Diluent CytoSelect™ Cell Dose Curve FBS Medium (mL) Agar Matrix Solution (mL) (mL) Solution (mL) 1.25 0.45 0.55 0.25 2.5 0.625 0.225 0.275 0.125 1.25 Table
        7. Preparation of Cell Dose Curve Solution 7
        8. Immediately dispense 125 μL of Cell Dose Curve Solution into the wells of the 96-well plate, already containing the cell serial dilution (from step 5).
        9. Add 125 μL of 1X Matrix Solubilization Buffer to each well. Pipette each well 10-12 times to mix thoroughly.
        10. Transfer 100 μL of the mixture to a 96-well microtiter plate.
        11. Add 10 μL of MTT Solution to each well. Pipette each well 7-10 times to ensure a homogeneous mixture.
        12. Incubate the plate for 2-4 hours at 37 °C and 5 % CO2. Note: Under the microscope, a purple precipitate should be visible within the cells.
        13. Add 100 μL of Detergent Solution to each well.
        14. Incubate the plate in the dark for 2-4 hours at room temperature.
        15. Pipette each well 7-10 times to ensure a homogeneous mixture.
        16. Measure the absorbance at 570 nm in a 96-well microtiter plate reader.

    Calculation of Results
    1. Compare OD570nm values with the Cell Dose Curve and extrapolate the cell concentration.
    2. Calculate the Total Transformed Cell Number/Well Total Transformed Cells/Well = cells/mL x 0.050 mL/well For example: If you extrapolate your OD570nm value from your cell dose curve and determine you have 500,000 cells/mL in your sample. Total Transformed Cells/Well = 500,000 cells/mL x 0.050 mL/well = 25,000 cells/well
    Restrictions
    For Research Use only
  • Storage
    4 °C
    Storage Comment
    Store all components at 4°C.
  • Kato, Mukudai, Motohashi, Ito, Kamoshida, Shimane, Kondo, Shirota: "Opposite effects of tumor protein D (TPD) 52 and TPD54 on oral squamous cell carcinoma cells." in: International journal of oncology, Vol. 50, Issue 5, pp. 1634-1646, (2017) (PubMed).

    Zheng, Yamamoto, Ishii, Sang, Hamashima, Van De, Nishizono, Inoue, Mori, Sasahara: "Glioma-Derived Platelet-Derived Growth Factor-BB Recruits Oligodendrocyte Progenitor Cells via Platelet-Derived Growth Factor Receptor-α and Remodels Cancer Stroma." in: The American journal of pathology, Vol. 186, Issue 5, pp. 1081-91, (2016) (PubMed).

    Wei, Lin, Wu, Chen, Li, Chen, Chou, Chen, Liang, Chien, Shiue: "A microtubule inhibitor, ABT-751, induces autophagy and delays apoptosis in Huh-7 cells." in: Toxicology and applied pharmacology, Vol. 311, pp. 88-98, (2016) (PubMed).

    Meador, Jin, de Stanchina, Nebhan, Pirazzoli, Wang, Lu, Vuong, Hutchinson, Jia, Chen, Eisenberg, Ladanyi, Politi, Zhao, Lovly, Cross, Pao: "Optimizing the sequence of anti-EGFR-targeted therapy in EGFR-mutant lung cancer." in: Molecular cancer therapeutics, Vol. 14, Issue 2, pp. 542-52, (2015) (PubMed).

    Akl, Ayoub, Ebrahim, Mohyeldin, Orabi, Foudah, El Sayed: "Araguspongine C induces autophagic death in breast cancer cells through suppression of c-Met and HER2 receptor tyrosine kinase signaling." in: Marine drugs, Vol. 13, Issue 1, pp. 288-311, (2015) (PubMed).

    Joshi, Jeon, Laganà, Middleton, Secchiero, Garofalo, Croce: "MicroRNA-148a reduces tumorigenesis and increases TRAIL-induced apoptosis in NSCLC." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 112, Issue 28, pp. 8650-5, (2015) (PubMed).

    Suman, Kurisetty, Das, Vadodkar, Ramos, Lakshmanaswamy, Damodaran: "Activation of AKT signaling promotes epithelial-mesenchymal transition and tumor growth in colorectal cancer cells." in: Molecular carcinogenesis, Vol. 53 Suppl 1, pp. E151-60, (2014) (PubMed).

    Suman, Das, Reddy, Nyakeriga, Luevano, Konwar, Pahari, Damodaran: "The pro-apoptotic role of autophagy in breast cancer." in: British journal of cancer, Vol. 111, Issue 2, pp. 309-17, (2014) (PubMed).

    Bard-Chapeau, Gunaratne, Kumar, Chua, Muller, Bard, Blackstock, Copeland, Jenkins: "EVI1 oncoprotein interacts with a large and complex network of proteins and integrates signals through protein phosphorylation." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 110, Issue 31, pp. E2885-94, (2013) (PubMed).

    Takezawa, Pirazzoli, Arcila, Nebhan, Song, de Stanchina, Ohashi, Janjigian, Spitzler, Melnick, Riely, Kris, Miller, Ladanyi, Politi, Pao: "HER2 amplification: a potential mechanism of acquired resistance to EGFR inhibition in EGFR-mutant lung cancers that lack the second-site EGFRT790M mutation." in: Cancer discovery, Vol. 2, Issue 10, pp. 922-33, (2012) (PubMed).

    Li, Yazawa, Kondo, Mukudai, Sato, Kurihara, Kamatani, Shintani: "The root bark of Paeonia moutan is a potential anticancer agent in human oral squamous cell carcinoma cells." in: Anticancer research, Vol. 32, Issue 7, pp. 2625-30, (2012) (PubMed).

    Itamochi, Oishi, Shimada, Sato, Uegaki, Naniwa, Sato, Nonaka, Terakawa, Kigawa, Harada: "Inhibiting the mTOR pathway synergistically enhances cytotoxicity in ovarian cancer cells induced by etoposide through upregulation of c-Jun." in: Clinical cancer research : an official journal of the American Association for Cancer Research, Vol. 17, Issue 14, pp. 4742-50, (2011) (PubMed).

    Kang, Lee, Yoon, Park, Choi, Min: "Phospholipase D1 drives a positive feedback loop to reinforce the Wnt/beta-catenin/TCF signaling axis." in: Cancer research, Vol. 70, Issue 10, pp. 4233-42, (2010) (PubMed).

  • Background
    Tumor sensitivity assays are intended to help predict the sensitivity of various tumors to chemotherapeutic agents, with the intent of identifying the most effective treatment with the fewest side effects. With this information, physicians can devise tailor made chemotherapy regiments and eliminate ineffective drugs, sparing patients of unnecessary toxicity. Ideally, an in vitro tumor sensitivity assay must be reliable, sensitive, and resemble the 3-D, in vivo environment (such as culturing in collagen gel or soft agar). Traditionally, the soft agar colony formation assay is a common method to monitor anchorage- independent growth, which measures proliferation in a semisolid culture media after 3-4 weeks by manual counting of colonies.
You are here:
Support