Biolegend, 426803, Cyto-Fast™ Fix/Perm Buffer Set, 300tests

Cyto-Fast™ Fix/Perm Buffer Set is composed of Cyto-Fast™ Fix/Perm Solution and Cyto-Fast™ Perm Wash Solution (10X). It is designed for fixation and permeabilization of mammalian cells for intracellular staining such as cytokines and other cytoplasmic molecules.
300tests
Storage & Handling: Upon receipt, store at 2-8°C.
Application: ICFC - Quality tested
Recommended Usage: Procedure for staining in round-bottom 5mL tubes: Dilute the Cyto-Fast™ Perm Wash solution (10X) to 1X using deionized water Aliquot 100 µL of cells (2 x 105-1 x 106) of interest into a 12 x 75 mm tube Add 150 µL of Cyto-Fast™ Fix/Perm Solution and mix Incubate for 20 minutes at room temperature Add 1 mL of 1X Cyto-Fast™ Perm Wash solution Centrifuge at 350 xg for 5 minutes, discard supernatant Repeat steps 5 - 6 Stain cells with optimal concentration of intracellular antibodies. Prepare antibodies in 1X Cyto-Fast™ Perm Wash Solution, in 100 µL total volume Incubate 20 minutes at room temperature in the dark Wash cells with 1 mL of 1x Cyto-Fast™ Perm Wash solution Centrifuge at 350 xg for 5 minutes, discard supernatant Add 1 mL of Cell Staining Buffer (Cat. No. 420201) Centrifuge at 350 xg for 5 minutes, discard supernatant Resuspend the cells in 300 µL of Cell Staining Buffer Acquire samples on a flow cytometer.Procedure for staining in 96-well U-bottom plates: Dilute the Cyto-Fast™ Perm Wash solution (10X) to 1X using deionized water Aliquot 100 µL of cells (2 x 105-1 x 106) of interest into individual wells in a 96-well U-bottom plate Centrifuge at 350 xg for 5 minutes, discard supernatant, and loosen cell pellet. Add 100 µL of Cyto-Fast™ Fix/Perm Solution and mix Incubate for 20 minutes at room temperature Add 100 µL of 1X Cyto-Fast™ Perm Wash solution Centrifuge at 350 xg for 5 minutes, discard supernatant Repeat steps 6 - 7 by adding 200 µL of 1X Cyto-Fast™ Perm Wash solution Stain cells with optimal concentration of intracellular antibodies. Prepare antibodies in 1X Cyto-Fast™ Perm Wash Solution, in 100 µL total volume Incubate 20 minutes at room temperature in the dark Wash cells by adding 100 µL of 1x Cyto-Fast™ Perm Wash solution Centrifuge at 350 xg for 5 minutes, discard supernatant Add 200 µL of Cell Staining Buffer (Cat. No. 420201) Centrifuge at 350 xg for 5 minutes, discard supernatant Resuspend the cells in 200 µL of Cell Staining Buffer Acquire samples on a flow cytometer.Addional Notes Cells can be bulk fixed. To bulk fix the cells, add 1.5 mL of Cyto-Fast™ Fix/Perm Solution for each 1 x 107 cells and incubate for 15-20 minutes at room temperature. Staining for surface markers can be performed prior to fix/perm. Follow the Cell Surface Flow Cytometry Staining Protocol. Proceed to follow the Cyto-Fast™ Fix/Perm Buffer Set protocol outlined above after primary (and/or secondary) surface antibody staining steps. If cells are to be immediately stained post-fix/perm, wash cells 2 x with 10 mL of 1X Cyto-Fast™ Perm Wash Solution. Aliquot 100µL of cells (2 x 105-1 x 106) of interest into a 12 x 75 mm tube and stain cells with optimal concentration of antibodies (continue with procedure listed above at step 9). If cells are to be stained at a later time post-fix/perm, wash cells with 10 mL of Cell Staining Buffer. Cells can be stored for up to one week in Cyto-Last™ Buffer (Cat. No. 422501). To stain cryopreserved cells, quickly thaw the cells by placing them in a 37°C water bath, wash with 10 mL of 1X Cyto-Fast™ Perm Wash Solution and centrifuge for 5 minutes at 200-300xg, discard supernatant. Re-suspend cells in 1X Cyto-Fast™ Perm Wash Solution for 15-20 minutes prior to staining. The Cyto-Fast™ Fix/Perm Buffer set (10x) may have crystallization or precipitation when it is stored at 2-8°C this is normal and does not affect the buffer performance. If heavy precipitation is observed after diluting to a 1X working solution, it can be filtered to clarify the solution.
Application Notes: Cyto-Fast™ Fix/Perm Buffer Set Contents: Cyto-Fast™ Fix/Perm Solution (50 mL) Cyto-Fast™ Perm Wash Solution 10X (100 mL)
Molecular Family: Cytokines/Chemokines
Gene ID: NA
Regulatory Status: RUO
Other Names: CytoFast