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ICC stimulation and staining


 

08/03/05
 
This protocol uses BD Bioscience intracellular kit (Cat # 555028)

  • Cell harvest/ Stimulation:
    TIL:  Harvest 10mm tumor and process through metal screen.  Pass cells through a 100um filter followed by 40um filter and resuspend in 5ml M.  Plate 1ml in a 24 well plate and add 1ul peptide.  For PMA/iono add 2ul PMA (50uM) and 5ul Ionomycin (100uM). 
     
    Splenoctyes:  Harvest spleens and plate 1x106 cells/well (in 100ul M) in a 96 well flat bottom plate.  Stimulate with a 1:1000 dilution of peptide.  For PMA/iono add 2ul 50uM PMA/ml and 5ul 100um Iono/ml.
     
    T cell clone:  Plate 5x105 T cells/well in a 96 well flat bottom plate with 1x106 splenocytes and 1:1000 peptide (in 100ul M total).  PMA/iono same as above
     
    Stimulate cells for 1hr at 37˚C.

  • Add equal volume of golgistop diluted 1:750 in M (1ml for TIL, 100ul for others).  Incubate for an additional 3-4hr.  
  • Harvest cells and transfer to a round bottom 96 well plate (For TIL transfer 1/2 of a 24 well into each 96 well).
  • Spin down cells and stain for cell surface markers in 50ul FCWB for 30 min at 4˚C. 
    BE SURE TO ADD Fc BLOCK TO TIL SAMPLES
  • Wash cells 2x with FCWB and add 50ul Cytofix/Cytoperm to each well.  Incubate 20 min at 4˚C.
  • Wash cells 2x with 1x Perm/Wash.  Stain for intracellular cytokines in 50ul Perm/Wash for 60 min at 4˚C.  (Overnight may be better and doesn’t cause too much background)
  • Wash cells 4x with Perm/Wash and 1x with FCWB and analyze by flow cytometry.