Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
B. Fixation
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
16% Formaldehyde (methanol free).
100% methanol.
Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
C. Permeabilization
D. Immunostaining
Collect cells by centrifugation and aspirate supernatant.
Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
Fix for 15 min at room temperature.
Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.
Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
Incubate 30 min on ice.
Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.
E. Optional DNA Dye
posted July 2009
revised June 2017
Product Usage Information
Application
Dilutions
Flow Cytometry
1:50
Storage: Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibodies. Protect from light. Do not freeze.
Specificity / Sensitivity
IDO (D5J4E™) Rabbit mAb recognizes endogenous levels of total IDO (IDO-1, INDO) protein. The antibody does not cross-react with IDO-2 (INDOL1). Some nonspecific staining of normal breast epithelium has been observed.
Species Reactivity: Human
Source / Purification
Monoclonal antibody is produced by immunizing animals with recombinant human IDO protein.
Product Description
This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated IDO (D5J4E™) Rabbit mAb #86630.
Aliquot desired number of cells into tubes or wells.
Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
Incubate for 1 hr at room temperature. Protect from light.
Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).
Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution#4087).
Incubate for at least 5 min at room temperature.
Analyze cells in DNA staining solution on flow cytometer.
原厂资料：
Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
B. Fixation
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
16% Formaldehyde (methanol free).
100% methanol.
Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
C. Permeabilization
D. Immunostaining
Collect cells by centrifugation and aspirate supernatant.
Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
Fix for 15 min at room temperature.
Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.
Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
Incubate 30 min on ice.
Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.
E. Optional DNA Dye
posted July 2009
revised June 2017
Product Usage Information
Application
Dilutions
Flow Cytometry
1:50
Storage: Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibodies. Protect from light. Do not freeze.
Specificity / Sensitivity
IDO (D5J4E™) Rabbit mAb recognizes endogenous levels of total IDO (IDO-1, INDO) protein. The antibody does not cross-react with IDO-2 (INDOL1). Some nonspecific staining of normal breast epithelium has been observed.
Species Reactivity: Human
Source / Purification
Monoclonal antibody is produced by immunizing animals with recombinant human IDO protein.
Product Description
This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated IDO (D5J4E™) Rabbit mAb #86630.
Aliquot desired number of cells into tubes or wells.
Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
Incubate for 1 hr at room temperature. Protect from light.
Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).
Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution#4087).
Incubate for at least 5 min at room temperature.
Analyze cells in DNA staining solution on flow cytometer.
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