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51995
Intracellular Flow Cytometry Kit (Triton™ X-100)
Buffers & Dyes
Buffer Kit

Intracellular Flow Cytometry Kit (Triton™ X-100) #51995

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Flow Cytometry Image 1: Intracellular Flow Cytometry Kit (Triton™ X-100)

To Purchase # 51995S
Product # Size Price
51995S
1 Kit  (100 tests) $ 179

Storage

All components in this kit are stable for at least 12 months when stored at the recommended temperature and left unused. Formaldehyde fixative should be used within one month after opening. Upon receipt, #13616 and #39487 should be stored at 4°C. Remaining components should be stored at room temperature.

Protocol

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Intracellular Flow Cytometry Kit (Triton™ X-100)

NOTE: Prior to using this kit, refer to the product webpage for antibodies of interest to determine whether they are validated for Flow Cytometry (F) assays using a Triton™ X-100 permeabilization protocol.

NOTE: When used in conjunction with fluorescent cellular dyes (including viability dyes, DNA dyes, etc.), please refer to the dye product page for recommended protocol. Visit www.cellsignal.com for a listing of flow cytometry validated cellular dyes.

A. Solutions and Reagents

Supplied Reagents:

  • 10X Wash Buffer, Phosphate Buffered Saline (PBS) (#12528): Dilute desired amount to a 1X working solution with reverse osmosis deionized (RODI) or equivalent grade water, mix. Store at 4°C and use within one month once diluted.
  • 4% Formaldehyde, methanol-free (#47746): Store at room temperature. Use within one month once opened.
  • Cell Permeabilization Buffer (Triton™ X-100) (#39487): Store at 4°C.
  • Flow Cytometry Antibody Dilution Buffer (#13616): Store at 4°C.

Additional Reagents (Not Supplied):

  • Red blood cell lysis buffer
  • Unconjugated or fluorochrome-conjugated primary antibodies
  • Fluorochrome-conjugated secondary antibodies (if applicable)
  • Cellular dyes (e.g., live-dead discrimination, DNA dyes, etc.), optional

NOTE: Not all extracellular antibodies will bind correctly after fixation and/or detergent permeabilization, so a small-scale experiment should be performed if you are unsure. Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation. The antibodies will remain bound to the target of interest during the fixation process. A wash step prior to fixation may be performed but is not necessary.

B. Fixation

NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.

  1. Pellet cells by centrifugation and remove supernatant.
  2. Resuspend cells in approximately 100 µl 4% Formaldehyde (#47746) per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
  3. Fix for 15 min at room temperature (20-25°C).
  4. Wash by centrifugation with excess 1X Wash Buffer. Discard supernatant containing formaldehyde in appropriate waste container. Repeat.
  5. Proceed to Permeabilization step, or store overnight in 1X PBS at 4°C.

C. Permeabilization

  1. If necessary, pellet cells by centrifugation and aspirate supernatant.
  2. Permeabilize cells by adding 100 µL Cell Permeabilization Buffer (Triton™ X-100) (#39487) per 1 million cells.
  3. Permeabilize for a minimum of 10 minutes.
  4. Proceed to Immunostaining step, or store cells overnight in 1X PBS at 4°C.

D. Immunostaining

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5 x 105 to 1 x 106 cells per assay.)
  2. Pellet cells by centrifugation and remove supernatant.
  3. Resuspend cells in 100 µl of diluted antibody or antibody conjugates, prepared in Flow Cytometry Antibody Dilution Buffer (#13616) at the recommended dilutions. See individual antibody datasheets or product webpage for recommended dilution, or determine via titration.
  4. Incubate for 1 hr at room temperature (20-25°C).
  5. Wash by centrifugation in excess 1X Wash Buffer. Discard supernatant. Repeat.
  6. If using a fluorochrome-conjugated primary antibody, resuspend cells in 200-500 µl 1X Wash Buffer and analyze on flow cytometer; for unconjugated primary antibodies, proceed to next step.
  7. Resuspend cells in fluorochrome-conjugated secondary antibody, diluted in Flow Cytometry Antibody Dilution Buffer (#13616) at the recommended dilution.
  8. Incubate for 30 min at room temperature (20-25°C).
  9. Wash by centrifugation in excess 1X Wash Buffer. Discard supernatant. Repeat.
  10. Resuspend cells in 200-500 µl 1X Wash Buffer and analyze on flow cytometer.

posted June 2019

revised June 2020

Protocol Id: 1844

Product Description

The Intracellular Flow Cytometry Kit (Triton™ X-100) provides the supporting reagents needed to preserve protein states and enable antibodies to bind intracellular targets, for flow cytometric analysis of cells in suspension. Detergent-based permeabilization provides access to the nucleus and other intracellular organelles, without affecting fluorophores of direct conjugates added prior to fixation. This kit contains sufficient reagents for 100 individual samples when following the included protocol.

IMPORTANT: Please refer to the antibody product page to determine if it is validated for use in Flow Cytometry (F) and for information regarding appropriate antibody dilution. Some primary antibodies may require methanol permeabilization, which will be noted on the datasheet. Methanol is not included in this kit.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST's products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST's Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Triton is a trademark of The Dow Chemical Company.