|Product Includes||Volume (with Count)|
|10X Wash Buffer, Phosphate Buffered Saline (PBS) 12528||1 x 125 ml|
|4% Formaldehyde, Methanol-Free 47746||1 x 20 ml|
|Methanol 13604||2 x 25 ml|
|Flow Cytometry Antibody Dilution Buffer 13616||1 x 25 ml|
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.
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.
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.
Additional Reagents (Not Supplied):
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.
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.
posted August 2018
Protocol Id: 1728
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.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.