Cyto-Fast™ Fix/Perm Buffer Set

Product Details

Storage & Handling

Upon receipt, store at 2-8°C.

Application

ICFC - Quality tested

Recommended Usage

Procedure for staining in round-bottom 5mL tubes:

1. Dilute the Cyto-Fast™ Perm Wash solution (10X) to 1X using deionized water
2. Aliquot 100 µl of cells (2 x 10⁵-1 x 10⁶) of interest into a 12 x 75 mm tube 
3. Add 150 µl of Cyto-Fast™ Fix/Perm Buffer and mix
4. Incubate for 20 minutes at room temperature
5. Add 1 ml of 1X Cyto-Fast™ Perm Wash solution
6. Centrifuge at 350 xg for 5 minutes, discard supernatant
7. Repeat steps 5 - 6
8. Stain cells with optimal concentration of intracellular antibodies. Prepare antibodies in 1X Cyto-Fast™ Perm Wash Solution, in 100 µl total volume
9. Incubate 20 minutes at room temperature in the dark
10. Wash cells with 1 ml of 1x Cyto-Fast™ Perm Wash solution
11. Centrifuge at 350 xg for 5 minutes, discard supernatant
12. Add 1 mL of Cell Staining Buffer (Cat. No. 420201)
13. Centrifuge at 350 xg for 5 minutes, discard supernatant
14. Resuspend the cells in 300 µl of Cell Staining Buffer
15. Acquire samples on a flow cytometer.

Procedure for staining in 96-well U-bottom plates:

1. Dilute the Cyto-Fast™ Perm Wash solution (10X) to 1X using deionized water
2. Aliquot 100 µl of cells (2 x 10⁵-1 x 10⁶) of interest into individual wells in a 96-well U-bottom plate 
3. Centrifuge at 350 xg for 5 minutes, discard supernatant, and loosen cell pellet.
4.  Add 100 µl of Cyto-Fast™ Fix/Perm Buffer and mix
5. Incubate for 20 minutes at room temperature
6. Add 100 µl of 1X Cyto-Fast™ Perm Wash solution
7. Centrifuge at 350 xg for 5 minutes, discard supernatant
8. Repeat steps 6 – 7 by adding 200 µl of 1X Cyto-Fast™ Perm Wash solution
9. Stain cells with optimal concentration of intracellular antibodies. Prepare antibodies in 1X Cyto-Fast™ Perm Wash Solution, in 100 µl total volume
10. Incubate 20 minutes at room temperature in the dark
11. Wash cells by adding 100 µl of 1x Cyto-Fast™ Perm Wash solution
12. Centrifuge at 350 xg for 5 minutes, discard supernatant
13. Add 200 µl of Cell Staining Buffer (Cat. No. 420201)
14. Centrifuge at 350 xg for 5 minutes, discard supernatant
15. Resuspend the cells in 200 µl of Cell Staining Buffer
16. 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 Buffer 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. 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 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)

Antigen Details

Molecular Family

Cytokines/Chemokines

Gene ID

NA

Documentation

• Technical data sheet
• Certificate of Analysis
• Material Safety Data Sheet

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Certificate of Analysis