Flow Cytometry can be used to investigate diverse intracellular Staining Protocol together with phosphorylated signaling proteins and cytokines. Moreover, the Cytokines and other secreted protocols can be detected by means of flow Cytometry in activated cells with the resource of secretion inhibitors, which include brefeldin A etc. These units save you the export of newly synthesized proteins. For experimental remedies with stimulation durations of as much as 5-6 hours, the secretion inhibitor may be present throughout the whole incubation duration. If the stimulation duration is longer than six hours, the secretion inhibitor needs to be brought for most effective the last three hours of the incubation.
There are abundance variables that need to be optimized for individual flow Cytometry experiments which include antibody incubation time and more. This permeabilization remedy lets in the antibody to bypass thru the plasma membrane into the cell interior, whilst keeping the morphological traits used to sort the cells. The name of commonly used detergents consists of Triton® X-a hundred, saponin, or Tween® 20.
Reagents Needed for Staining Intracellular Procedure
- Flow Cytometry Fixation Buffer
- Detection Antibodies
- Flow Cytometry Fixation / Permeabilization Buffer I
- PBS (1X): 0.137 M NaCl, 0.05 M NaH2PO4, pH 7.4 or Hank’s Balanced Salt Solution (HBSS; 1X)
- Isotype Control Antibodies
Materials needed for Staining Intracellular Procedure
- FACS™ Tubes
- Centrifuge
- Vortex
- Pipette Tips and Pipettes
Procedure
- First of all, harvest the cells and then clean it 2 times with 2 mL of PBS or HBSS, and then decanting buffer from pelleted cells.
- Then, aliquot up to 1 x 106 cells/100 μL into FACS tubes and combine 0.5 mL of cold Flow Cytometry Fixation Buffer and vortex. Then stay at room temperature for fifteen minutes.
- In this step, centrifuge cells and decant the Fixation Buffer and then clean the cells 2 times again with PBS or HBSS.
- Input or add 10 μL of conjugated antibody and vortex and then again stay these cells for half an hour at room temperature in the dark.
- In the last step, again clean cells 2 times with Flow Cytometry Permeabilization / Wash Buffer I as did in step 3.
It should be noted that in any circumstances if an unconjugated primary antibody changed into used, incubation with the secondary antibody need to occur now. Dilute the secondary antibody in Flow Cytometry Permeabilization or Wash Buffer I, beginning with the concentration cautioned inside the product datasheet. Then stay it for half an hour within the dark and as did in step 3.
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