Haynes:ICCSaponin: Difference between revisions
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* '''Paraformaldehyde''' (EMS Cat # 15714) - 32% Solution, EM Grade, MeOH-free | * '''Paraformaldehyde''' (EMS Cat # 15714) - 32% Solution, EM Grade, MeOH-free | ||
** Dilute to final concentration of 1% in 1X PBS just before each use. | ** Dilute to final concentration of 1% in 1X PBS just before each use. | ||
* FACS Buffer - PBS with 1% FBS (stored at 4°C) | * '''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) | * '''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. | ** Dilute to final concentration of 0.2% in FACS Buffer just before each use. | ||
Revision as of 08:58, 7 June 2013
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
- Obtain an ice bucket. Aliquot 4 mL FACS buffer (per sample) into a conical tube. Chill on ice.
- Harvest ~2x106 cells (~2 wells of adherent mammalian cells from a 6-well plate) using the standard trypsinization procedure.
- Collect the cells in growth medium and transfer to a 15 mL conical tube.
- Pellet the cells at room temperature at 1000 rpm for 3 min.
- 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.
- 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
- Make 1% Paraformaldehyde Solution
- 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.
