Cyto-Fast™ Fix/Perm Buffer Set

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CytoFast
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Cyto-Fast_Fix_Perm_Buffer_Set_110718
PMA + ionomycin stimulated (3 hours) human peripheral blood lymphocytes (in the presence of monensin) were fixed and permeabilized with Cyto-Fast™ Fix/Perm Buffer Set. Cells were then stained with CD3 (UCHT1) APC and isotype control PE (left panel), IL-2 (clone MQ1-17H12) PE (middle panel), or IFN-γ (clone 4S.B3) PE (right panel).
  • Cyto-Fast_Fix_Perm_Buffer_Set_110718
    PMA + ionomycin stimulated (3 hours) human peripheral blood lymphocytes (in the presence of monensin) were fixed and permeabilized with Cyto-Fast™ Fix/Perm Buffer Set. Cells were then stained with CD3 (UCHT1) APC and isotype control PE (left panel), IL-2 (clone MQ1-17H12) PE (middle panel), or IFN-γ (clone 4S.B3) PE (right panel).
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426803 300 tests £158
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Description

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 permeabiliztion of mammalian cells for intracellular staining such as cytokines and other cytoplasmic molecules.

Product Details
Technical Data Sheet (pdf)

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.


Note: 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. If cells are to be immediately stained, 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, 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). If cells are to be kept for long term storage, freeze cells at -80°C in a 10% DMSO containing medium. (E.g. 90% FCS and 10% DMSO). Cells may be kept at -80°C for a prolonged period of time. To thaw the cells place 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.

 

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 5 – 6 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.
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

Related FAQs

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Go To Top Version: 2    Revision Date: 11/29/2018

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