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13593
Intracellular Flow Cytometry Kit (Methanol)
Buffers & Dyes

Intracellular Flow Cytometry Kit (Methanol) #13593

Product Includes Volume (with Count)
10X Wash Buffer, Phosphate Buffered Saline (PBS) 12528 1 x 125 ml
4% Formaldehyde, Methanol-Free 1 x 20 ml
Methanol 2 x 25 ml
Flow Cytometry Antibody Dilution Buffer 1 x 25 ml

#13593 Intracellular Flow Cytometry Kit (Methanol)

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 methanol 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/flowdyes for a listing of flow cytometry validated cellular dyes.

A. Solutions and Reagents

Supplied Reagents:

  1. 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. 1X PBS is pH 8.0.
  2. 4% Formaldehyde, methanol-free (#47746)
  3. 100% Methanol (#13604): Keep at room temperature for long-term storage. Chill to -20°C before use.
  4. Flow Cytometry Antibody Dilution Buffer (#13616): Store at 4°C. Formulation: 1X PBS, 0.5% BSA, 0.01% sodium azide, pH 8.0.

Additional Reagents (Not Supplied):

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

NOTE: Fluorescent proteins such as PE and APC are damaged by methanol. Any antibodies conjugated to fluorescent proteins should be added after the methanol permeabilization step to avoid loss of fluorescence. Not all extracellular antibodies will bind correctly after methanol permeabilization, so a small-scale experiment should be performed if you are unsure.

B. Fixation

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

NOTE: Fixable viability dyes such as Ghost Dye™ Violet 510 Viability Dye #59863 should be added prior to fixation, following the product protocol. Proceed with fixation once excess dye has been removed.

NOTE: 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.

  1. Pellet cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 1-2 mL 4% formaldehyde, mix well to dissociate pellet and prevent cross-linking of individual cells.
  3. Fix for 15 min at room temperature (20-25°C).
  4. Proceed to Permeabilization step.
    1. Alternatively, cells may be stored overnight in PBS, or antibodies against methanol-sensitive extracellular epitopes may be added. To remove formaldehyde, wash by centrifugation with excess 1X PBS. Discard supernatant containing formaldehyde in appropriate waste container. Store cells at 4°C in PBS, or add extracellular antibodies following section D (Immunostaining).

C. Permeabilization

  1. Permeabilize cells by adding ice cold 100% Methanol drop-wise to pre-chilled cells, while gently vortexing, to a final concentration of 90% Methanol (e.g. 1 ml of fixed cells in formaldehyde + 9 ml of Methanol). Alternatively, remove formaldehyde or PBS by centrifugation and resuspend in ice cold 90% Methanol (v/v in 1X PBS) as described above.
  2. Permeabilize for a minimum of 10 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% Methanol.

D. Immunostaining

  1. Aliquot desired number of cells into tubes or wells.
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted antibody or antibody conjugates, prepared in 1X Incubation Buffer 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 PBS. Discard supernatant. Repeat.
  6. If using a fluorochrome-conjugated primary antibody, resuspend cells in 500 µl 1X PBS and analyze on flow cytometer; for unconjugated primary antibodies, proceed to next step.
  7. Resuspend cells in fluorochrome-conjugated secondary antibody, diluted in 1X Incubation Buffer at the recommended dilution.
  8. Incubate for 30 min at room temperature (20-25°C).
  9. Wash by centrifugation in excess 1X PBS. Discard supernatant. Repeat.
  10. Resuspend cells in 500 µl 1X PBS and analyze on flow cytometer.

posted August 2018

The Intracellular Flow Cytometry Kit provides the supporting reagents needed to preserve protein states and enable antibodies to bind intracellular targets, for flow cytometric analysis of cells in suspension. 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 detergent permeabilization, which will be noted on the datasheet. Detergent is not included in this kit.

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 should be stored at 4°C. Remaining components should be stored at room temperature.

  1. Red blood cell lysis buffer
  2. Unconjugated or conjugated primary antibody
  3. Fluorochrome-conjugated secondary antibody (if applicable)

    NOTE: When using any primary or secondary antibody for the first time, titrate the antibody to determine which dilution allows for the strongest specific signal with the least background for your sample.

  4. Cellular dyes (e.g., live-dead discrimination, DNA dyes, etc.), optional
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

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