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The reconstituted human PBMCs can be used for intracellular flow cytometry assays. They can also be used as a process control to ensure effective permeabilization when performing intracellular staining using True-Phos™ Perm Buffer.
This product contains human cells, a potentially biohazardous material. Blood used in preparation of these samples was tested and found to be negative, using FDA approved methods, for antibody and nucleic acid testing against Human Immunodeficiency virus (HIV), Hepatitis B and C virus and HTLV (Human T-Lymphotrophic virus I and II), surface antigen for Hepatitis B (HBsAg) virus and syphilis and West Nile Virus. Biological tests are not 100% accurate. Use standard precautions when handling and treat as if the product is capable of transmitting disease. When handling or disposing, follow precautions described in CDC and FDA recommendations and OSHA Bloodborne Pathogen guidelines.
- Kit Contents
Cat. No. 426601
- 2 vials of Veri-Cells™ Phospho PBMC (MAPK/ERK pathway) Vehicle Treated Cells
- 2 vials of Veri-Cells™ Phospho PBMC (MAPK/ERK pathway) Stimulated Cells
- 1 vial of Veri-Cells™ Reconstitution Buffer A
Cat. No. 426603
- 10 vials of Veri-Cells™ Phospho (MAPK/ERK pathway) Vehicle Treated Cells
- 10 vials of Veri-Cells™ Phospho (MAPK/ERK pathway) Stimulated Cells
- 5 vials of Veri-Cells™ Reconstitution Buffer A
- Each kit contains an equal number of vials of unstimulated (vehicle treated) and stimulated Veri-Cells™ Phospho PBMC (MAPK/ERK pathway) cells (2 vials each for 10 tests, and 10 vials each for 50 tests), as well as Veri-Cells™ Buffer A (1 vial for 10 tests and 5 vials for 50 tests). Each vial of Veri-Cells™ Phospho PBMC (MAPK/ERK pathway) contains approximately 1 x 106 lyophilized human peripheral blood mononuclear cells (PBMC).
- The Veri-Cells™ Phospho PBMC (MAPK/ERK pathway) Kit is prepared by stimulating human PBMCs with the Cell Activation Cocktail (Cat. No. 423301) for 15 minutes. The lyophilized cells can be reconstituted with Veri-Cells™ Buffer A and permeablized with True-Phos™ Perm Buffer (Cat. No. 425401).
- Storage & Handling
- Store the kit at 2°C - 8°C upon receipt. Do not open the sealed pouch until ready to use. The cells may be used for up to 5 days if properly stored at 2°C - 8°C in the buffer provided and processed according to the defined protocol.
ICFC - Quality tested
- Recommended Usage
50 µL per test (after reconstitution with 325 µL)
- Application Notes
The Veri-Cells Phospho PBMC (MAPK/ERK pathway) Kit can be used as both positive and negative staining controls for members of the RAS mitogen-activated kinase (MAPK/ERK) pathway such as phospho-S6, phospho-p38 and phospho-ERK 1/2. Additional intracellular targets may be detectable outside of those detailed on the CoA.
Once reconstituted, the Veri-Cells™ Phospho PBMC (MAPK/ERK pathway) are processed and stained per the protocol detailed below. Deviations from the use of these reagents and procedures have not been assessed and may affect performance.
Protocol for using Veri-Cells™ Phospho PBMC (MAPK/ERK Pathway):
- One hour prior to processing, place a bottle of True-Phos™ Perm Buffer (Cat. No. 425401) in a -20°C freezer.
- Remove a vial of Veri-Cells™ Phospho PBMC (MAPK/ERK pathway) Stimulated Cells and a vial of Veri-Cells™ Phospho PBMC (MAPK/ERK pathway) Vehicle Treated Cells from the refrigerator and warm to room temperature for 5 minutes.
- Add 325µl of Veri-Cells™ Buffer A included with the kit to each vial.
- Ensure the entire cell pellet is dissolved and incubate for 10 minutes (do not vortex).
- 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.
- Add 2ml of Cell Staining Buffer (Cat. No. 420201) and centrifuge the tubes at 1000 x g for 5 minutes at room temperature. Discard the supernatant.
- Wash two more times with 2ml of Cell Staining Buffer at 1000 x g for 5 minutes. Discard the supernatant.
- Vortex the cells to loosen the pellet. Note: Significant cell loss will occur during permeabilization if the pellet is not fully dissociated.
- Permeabilize the cells by slowly adding 2.5ml of chilled True-Phos™ Perm Buffer while vortexing.
- Incubate the suspension at -20°C for 1 hour. Note: The cells are stable for 5 days if stored at -20°C in the True-Phos™ buffer.
- Centrifuge the tube at 1000 x g for 5 minutes at room temperature. Discard the supernatant.
- Wash two times with 2ml of Cell Staining Buffer at 1000 x g for 5 minutes. Discard the supernatant.
- If the entire vial has been processed, Add 250µl of Cell Staining Buffer (50µl / Test) and vortex to resuspend.
- Add 50µl of the cell suspension to intracellular and/or surface antibodies and vortex to mix. Incubate in the dark at room temperature for 30 minutes.
- Wash the cells with 2ml of wash Cell Staining Buffer two times at 1000 x g for 5 minutes at room temperature. Discard the supernatant.
- Resuspend the stained cells in an appropriate volume of Cell Staining Buffer for flow cytometric analysis.
Please use our CoA Look-Up Tool to find the expected lot-specific frequencies of certain cell populations included within each lot of Phospho PBMC (MAPK/ERK Pathway). Percent positive is based on gates set on unstained cells. The Certificate of Analysis specific values are provided only as a reference. Variability in antibodies used, instruments, staining conditions and other factors may influence end-user values. It is recommended that individual laboratories establish its own control values and ranges for each parameter based on laboratory specific conditions and protocols.
- Biology Area
- Molecular Family
- Gene ID