Protocol for using Veri-Cells™

Once reconstituted, the Veri-Cells™ products are processed and stained per the protocol details below. Deviations from the use of these reagents and procedures have not been assessed and may affect performance.

 

Reagent List

Protocol for using Veri-Cells™ products (See further below for phospho and activated PBMC protocols)


 

  1. Prior to reconstitution, warm lyophilized vial(s) of Veri-Cells™ at room temperature for 5 minutes.
    Note: For 5 test size vials, use 325 µl of Veri-Cells™ Buffer A. For 25 test size vials, use 1.3 ml of Veri-Cells™ Buffer A. For sizing, check the product label or refer to the section Kit Components on the Technical Data Sheet.
  2. Aliquot the required amount of cells into test tubes (we recommend using 50 µl). Stain with antibodies using our recommended surface or intracellular staining protocols. Cell Staining Buffer (Cat. No. 420201) is recommended for washing and analysis steps. The cells are ready for acquisition.
    Note: To monitor non-specific staining, we recommend using appropriate isotype controls.

 

Protocol for using Veri-Cells™ Phospho PBMC (MAPK/ERK Pathway) Kit


 

  1. Prior to processing, chill a bottle of True-Phos™ Perm Buffer (Cat. No. 425401) in a -20°C freezer.
  2. Remove a vial each of Veri-Cells™ Phospho PBMC (MAPK/ERK pathway) Stimulated Cells and Vehicle Treated Cells from the refrigerator and warm to room temperature for 5 minutes.
  3. Add 325 µl of Veri-Cells™ Buffer A, included with the kit, to each vial.
  4. Ensure the entire cell pellet is dissolved and incubate for 10 minutes (do not vortex).
  5. Gently mix the vials to resuspend the cells and transfer all or the required volume of cells to separate FACS tubes.
    Note: The reconstituted cells are stable in the buffer provided for up to 5 days when stored at 4°C.
  6. Add 2 ml of Cell Staining Buffer (Cat. No. 420201) and centrifuge the tubes at 1000 x g for 5 minutes at room temperature. Discard the supernatant.
  7. Wash two more times with 2 ml of Cell Staining Buffer at 1000 x g for 5 minutes. Discard the supernatant.
  8. Vortex the cells to loosen the pellet.
    Note: Significant cell loss will occur during permeabilization if the pellet is not fully dissociated.
  9. Permeabilize the cells by slowly adding 2.5 ml of chilled True-Phos™ Perm Buffer while vortexing.
  10. Incubate the suspension at -20°C for 1 hour.
    Note: The cells are stable for 5 days if stored at -20°C in True-Phos™ buffer.
  11. Centrifuge the tube at 1000 x g for 5 minutes at room temperature. Discard the supernatant.
  12. Wash two times with 2 ml of Cell Staining Buffer at 1000 x g for 5 minutes. Discard the supernatant.
  13. If the entire vial has been processed, add 250 µl of Cell Staining Buffer (50 µl/test) and vortex to resuspend.
  14. Add 50 µl of the cell suspension to intracellular and/or surface antibodies and vortex to mix. Incubate in the dark at room temperature for 30 minutes.
  15. Wash the cells with 2 ml of Cell Staining Buffer two times at 1000 x g for 5 minutes at room temperature. Discard the supernatant.
  16. Resuspend the stained cells in an appropriate volume of Cell Staining Buffer for flow cytometric analysis. 
    Note: To monitor non-specific staining, we recommend using appropriate isotype controls.

 

Protocol for using the Veri-Cells™ Activated (Cytokine) PBMC Kit


 

  1. Prior to reconstitution, warm lyophilized vial(s) of Veri-Cells™ Activated (Cytokine) PBMC at room temperature for 5 minutes.
  2. Add 325 µl of Veri-Cells™ Buffer A to the cell pellet. Replace the rubber stopper and mix gently for 5 seconds. Allow at least 5 minutes for the cells to rehydrate prior to staining. Each vial contains enough reagent for 5 tests.
  3. Optional, fix and permeabilize the cells (Cat. Nos. 420801 and 421002 are recommended)
    Note: Veri-Cells™ Activated (Cytokine) PBMC do not need to be fixed and permeabilized to achieve cytokine staining. Optional fix/perm steps can be added here to serve as a process control to match experimental sample treatments, if desired.
  4. Aliquot the required amount of cells into tubes (we recommend using 50 µl). Stain with antibodies using our Cell Surface Immunofluorescence Staining Protocol . The cells are ready for acquisition. Cell Staining Buffer (Cat. No. 420201) is recommended for washing Veri-Cells™ Activated (Cytokine) PBMC.
  5. Wash the cells twice with 2 ml of Cell Staining Buffer. The cells are ready for acquisition. 
    Note: To monitor non-specific staining, we recommend using appropriate isotype controls.

 

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