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Yale Flow Cytometry
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INFORMATION FOR

Intracellular Cytokine Staining Procedure

Prepare Media from stocks.

Stocks

  • Ionomycin - 2.5 mg/ml in DMSO - stored -80°C
  • 10µl stock — 40µl Clone Medium = diluted ionomycin (500µg/ml)
  • Calbiochem cat #542400
  • PMA - 2.5 mg/ml in DMSO - stored -80°C
  • 10µl stock — 490µl Clone Medium = diluted PMA (50µg/ml)
  • Calbiochem cat #407952
  • Monensin - 2mM in ETOH - stored -20°C - Sigma cat#M5273
  • Good IX PBS - stored room temperature - lab prepared
  • FBS - stored -20°C - Gemini Bio Products cat #100-106
  • NaN3 - 20% - stored room temperature - lab prepared
  • CytoFix/CytoPerm Kit: - stored 4°C - Pharmingen cat#2075KK
  • DNase — 1mg/ml in PBS — stored -20°C — Sigma cat#D5025
  • EMA — stored -20°C - Molecular Probes cat#E1374
  • Para-formaldehyde - 10% - stored -20°C - lab prepared

Clone Medium

  • RPMI 1640 w/o L-glutamine (500ml)
  • 10% FCS (50ml)
  • 2mM L-glutamine (5ml)
  • 10mM Hepes (5ml)
  • 25ug/ml gentamicin (1ml)
  • 50uM 2-ME (0.5ml)
  • 2U/ml IL-2 (0.85ml)

Stock Solutions

  • FCS (HyClone)
  • L-glutamine - 200mM
  • Hepes buffer - 1M
  • gentamicin sulfate - 12.5mg/ml
  • 2-ME - 50mM
  • IL-2 - 1200U/ml

Stimulating Medium

  • 50µl diluted ionomycin (50µg/ml)
  • 5µl diluted PMA (0.5µg/ml)
  • 445µl Clone Medium
  • 0.5ml total volume - use immediately

(Hint: Add 485 µl Clone Medium to ionomycin vial; add 490 µl Clone Medium to PMA vial, mix and transfer 5µl of diluted PMA to ionomycin vial yielding the final solution.)

Stain Medium

  • 1L Good 1X PBS
  • 30ml FBS (3%)
  • 2ml NaN3 stock (0.04%)
  • approx 1L total volume - store 4°C - 6 months

PBS-A

  • 1L Good 1X PBS
  • 2ml NaN3 stock (0.04%)
  • approx 1L total volume - store RT - 1 year

DNAse Solution

  • PBS-A containing 10% stock DNAse (0.1mg/ml)
  • (1ml per sample - make an extra dose)
  • Must be made fresh.

EMA Staining Mix

  • Stain Medium + 0.04% EMA
  • (250µl per sample - make an extra dose)
  • Must be made fresh - protect from light at all times.

CytoFix/CytoPerm: Pharmingen:

  • Cytofix/Cytoperm: pre-prepared
  • Perm/Wash Buffer: dilute 1:10 in ddH2O

Final Fixing Buffer:

  • 1ml para-formaldehyde stock (1%)
  • 9ml Stain Medium
  • 10ml total volume - use immediately - light sensitive

Isolation/Preparation of Spleen Cells

  • Collect spleens from mice and place each in a small petri dish containing 5ml Clone Medium.
  • Isolate lymphocytes using the syringe puncture method.
  • Wash and lyse rbcs with ACT.
  • Wash with Clone Medium and resuspend in 2ml Clone Medium.
  • Place exactly 1ml in one well of a 12 well plate.
  • Add an additional 1ml of Clone Medium to the well.
  • Can store spleen suspensions in 12 well plate overnight in refrigerator.
  • Use remaining spleen cells for phenotype FACS if desired - wash with Staining Buffer to remove Clone Medium.

Stimulation of Spleen Cells

Prepare Stimulating Medium.

  • Add 10µl Stimulating Mix per 1ml of cell suspension (ie 20µl/well).
    Note: final concentrations should be 5ng/ml PMA and 500ng/ml ionomycin.
  • Incubate at 37°C for 3 hours.
  • Add 1µl Monensin stock per 1ml cell suspension (ie 2µl/well).
    Note: final concentration should be 1µM Monensin.
  • **Remember to thaw out Th1 and Th2 positive cells to treat along with spleen cells in the following steps.**
  • Place cells into FACS tubes containing 1ml Stain Medium and centrifuge at 1200rpm for 5 min.
  • Resuspend in 1ml DNAse Solution.
  • Incubate in 37°C water bath for 10 minutes.
  • **Remove approximately 10ul of cells from one sample's cell pellet to be used for unstained and single color controls**
  • Prepare EMA staining mix - protect from light.
  • Resuspend each sample in 250µl EMA Staining Mix.
  • Incubate on ice PROTECTED FROM LIGHT for 15 minutes.
  • Expose to fluorescent light for 10 minutes - place on bench under bench-top fluorescent light - approx. 12-18 inches away.
  • Wash in 3ml Stain Medium.
  • Resuspend each pellet in 200µl CytoFix/CytoPerm solution.
  • Incubate in refrigerator or on ice PROTECTED FROM LIGHT for 20 minutes.
  • Wash 2 times in 1ml Perm/Wash Buffer.
  • Resuspend cell pellet in 50µl Perm/Wash Buffer X number of rxns.
  • Remove some cells for EMA single color control.
  • Distribute 50µl into individual tubes or wells.
  • Add antibody mixes to appropriate wells.
  • Incubate in refrigerator for 30-60 minutes.
  • Wash 2x with CytoPerm Wash Buffer.
  • Resuspend in 50µl Final Fixing Buffer.
  • Place cell suspension in labeled FACS tubes containing 100-200µl Stain Medium prior to running on FACSCalibur.