Introduction

Stimulation reagents, phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin A, and monensin are useful to activate transcription factors for intracellular signaling and production of cytokines of many different immune cell types. Brefeldin A solution is required for intracellular retention of signaling proteins and cytokines. Brefeldin A is an inhibitor of intracellular protein transport. Incubation of cells in culture with brefeldin A leads to blockade of protein transport to the Golgi complex (GC) and accumulation of proteins in the endoplasmic reticulum (ER). Addition of brefeldin A during the last hours of in vitro activation of cells results in enhanced detection of intracellular cytokines. If performing an immunoassay on secreted protein (e.g., ELISA, western or multiplex protein detection), cells do not require treatment with brefeldin A.


Materials

  1. T25 or T75 sterile flasks with vented caps (e.g., Nunc EasYFlask Cell Culture Flasks, T25, filter, Cat. No. 156367)
  2. RPMI 1640 medium (e.g., BenchStable RPMI 1640 Medium, Cat. No. A4192301)
  3. Fetal Bovine Serum (e.g., Gibco Fetal Bovine Serum, Cat. No. 26140079)
  4. Cell Stimulation Cocktail: phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin A, and monensin (e.g., Invitrogen Cell Stimulation Cocktail with or without protein transport inhibitors, Cat. No. 00-4975-93 or 00-4970-93)
  5. Brefeldin A solution (e.g., eBioscience Brefeldin A solution, Cat. No. 00-4506-51)
  6. Cell scraper (e.g., Nunc Cell Scrapers, Cat. No. 179693)


Procedure

  1. Using aseptic techniques under sterile conditions, isolate PBMC from whole blood (supplemental protocol A) or prepared lymphoid tissue (supplemental protocol B).
  1. Prepare complete RPMI 1640 medium by supplementing RPMI 1640 Medium with fetal bovine serum to a final concentration of 10%. Bring medium to 37°C.
  1. Resuspend cells to a concentration of 3 x 106 cells/mL in complete RPMI 1640 medium. Prepare the volume needed based on the flask size you will be using (T25 or T75).
  1. Prepare 2 culture flasks: one labeled ‘stimulated’ (activated) and the other as ‘non-stimulated’ (non-activated).
  1. Divide the cell solution (prepared in step 3) evenly into the prepared flasks.
  1. Add Cell Stimulation Cocktail at a concentration of 1X, 2 µL/mL into the stimulated (activated) flask.
  1. Add Brefeldin A solution at a concentration of 1X, 3 µL/mL to each of the stimulated and non-stimulated flasks.
  1. Cover both flasks with vented filtered caps, and incubate for 5 hours in 5% CO2 incubator at 37°C.
  1. Harvest cells with a cell scraper.

Protocol tip:

Use of ready-made fixation buffers can help improve detection of cytokines and transcription factors in flow cytometry applications.


Supplemental protocol A: isolation of PBMC from whole blood

Materials

  1. Phosphate buffered saline (e.g., Gibco PBS (10X), pH 7.4, Cat. No. 70011044)
  2. 15 mL or 50 mL conical tube (e.g., Nunc 15 mL conical sterile centrifuge tubes, Cat. No. 339650)
  3. Ficoll-Paque® density separation medium
  4. Flow cytometry staining buffer (e.g., eBioscience Flow Cytometry Staining Buffer, Cat. No. 00-4222-26)

Procedure

  1. Dilute blood sample at least 1:1 with PBS in a conical tube.
  1. Underlay the diluted sample with a volume of Ficoll-Paque® medium that is equal to the original sample volume.
  1. Centrifuge at 400 x g for 20 minutes at room temperature with the brake OFF.
  1. Harvest PBMC located at the interface of the PBS and Ficoll-Paque® medium layers into a fresh tube.
  1. Fill the tube with PBS to wash the cells.
  1. Centrifuge the cells at 300–400 x g for 4–5 minutes at 2–8°C. Discard supernatant.
  1. Resuspend the cell pellet in an appropriate volume of flow cytometry staining buffer or buffer of choice, and perform a cell count and viability analysis.
  1. Centrifuge cells as in step 6, and resuspend in appropriate volume of complete RPMI 1640 so that the final cell concentration is 3 x 106 cells/mL.

Note:

As cells will be cultured, perform all steps using aseptic technique, and use buffers that do not contain azide.

Protocol tip:

Significantly improve the accuracy of assessing cell health and concentration from freshly harvested peripheral blood mononuclear cells with automated counters. See detailed protocol on how to count PBMC using the Countess II FL Automated Cell Counter.


Supplemental protocol B: isolation of immune cells from lymphoid tissue

Materials

  1. 60 x 15 mm cell culture dish (e.g., Nunc Cell Culture Petri Dishes, Cat. No. 150340)
  2. Plastic 3-mL syringe or two frosted glass microscope slides
  3. Cell strainer (nylon mesh)
  4. 15 mL or 50 mL conical tube (e.g., Nunc 15 mL conical sterile centrifuge tubes, Cat. No. 339650)
  5. Flow cytometry staining buffer (e.g., eBioscience Flow Cytometry Staining Buffer, Cat. No. 00-4222-26)

Procedure

  1. Harvest tissue (spleen; thymus; lymph nodes) into a cell culture dish containing 10 mL of Flow Cytometry Staining Buffer or buffer of choice. Tease apart into a single-cell suspension by pressing with the plunger of a 3 mL syringe. Alternatively, mash tissue between the frosted ends of two microscope slides using 10 mL of Flow Cytometry Staining Buffer.
  1. Place a cell strainer on top of a 15-mL conical tube. Pass cells from the cell culture dish through the cell strainer to eliminate clumps and debris.
  1. Centrifuge cell suspension at 300–400 x g for 4–5 minutes at 2–8°C. Discard the supernatant.
  1. Resuspend the cell pellet in an appropriate volume of Flow Cytometry Staining Buffer or buffer of choice and perform a cell count and viability analysis.
  1. Centrifuge cells as in step 3, and resuspend in appropriate volume of complete RPMI 1640 medium to the final cell concentration is 3 x 106 cells/mL.

Note:

Mechanical disruption of lymphoid tissue is generally sufficient to release cells into a single-cell suspension.

Note:

As cells are to be cultured, perform all steps using aseptic technique, and use buffers that do not contain azide.