Haynes:ICCSaponin

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Intracellular Protein Staining with Saponin Permeabilization
Karmella Haynes 2013
Adapted from protocol by Jodene K. Moore, Harvard Systems Biology, 2013

Reagents:

• Paraformaldehyde (EMS Cat # 15714) - 32% Solution, EM Grade, MeOH-free
  • Dilute to final concentration of 1% in 1X PBS just before each use.
• FACS Buffer - PBS with 1% FBS (stored at 4°C)
• FB-S Saponin Staining/Wash Buffer (Sigma Catalog # S4521) - 10% stock (stored at 4°C)
  • Dilute to final concentration of 0.2% in FACS Buffer just before each use.

Protocol: This procedure is for a single sample. Scale as needed.

Harvest the cells

1. Obtain an ice bucket. Aliquot 4 mL FACS buffer (per sample) into a conical tube. Chill on ice.
2. Harvest ~2x10⁶ cells (~2 wells of adherent mammalian cells from a 6-well plate) using the standard trypsinization procedure.
3. Collect the cells in growth medium and transfer to a 15 mL conical tube.
4. Pellet the cells at room temperature at 1000 rpm for 3 min.
5. Aspirate off the growth medium, but leave enough to cover the pellet. Flick the tube to break up the pellet. Chill the cells on ice.
6. Do this step twice: Add 1 mL cold FACS buffer to the cells. Pellet the cells at room temperature at 1000 rpm for 3 min. Aspirate off the buffer, leaving enough to cover the pellet. Flick the tube to break up the pellet.

Paraformaldehyde Fixation

1. Make 1% Paraformaldehyde Solution
2. Add 1ml of 1% Paraformaldehyde Solution and incubate for 15 minutes on ice. (Longer incubations compromise the quality of CV’s in DNA histograms.)

• Wash 2X with cold FACS Buffer.

Saponin Permeabilization • Decant the last wash and resuspend pellet in remaining residual volume. • Resuspend cells in 1 ml FB-S, added slowly while flicking the sample. • Incubate at RT for 30 minutes. • Spin down. Decant. Resuspend cell in the residual volume of FB-S. Should be about 100ul.

Antibody Staining (All staining and washes are to be done in FB-S) • Incubate cells in 100ul final volume of antibody(ies), diluted to recommended concentration in FB-S, for 30 minutes on ice. • Wash cells 2X in cold FB-S. Decant Supernatant. • Resuspend in 500ul of FACS Buffer. • Analyze on Flow cytometer. (keep covered on ice until analysis; always have NEGATIVE control)

2nd Antibody Staining • Follow directions as above in Antibody Staining.