Haynes:ICCSaponin

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Reagents:
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REAGENTS:
* '''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.
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Protocol: This procedure is for a single experimental sample. Scale-up as needed.
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PROTOCOL<br>
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''This procedure is for a single experimental sample. Scale-up as needed.''
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<br>
'''Harvest the cells'''
'''Harvest the cells'''
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# 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.
# 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.
# Wash the cells 2x with cold FACS buffer: 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. Repeat this process.
# Wash the cells 2x with cold FACS buffer: 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. Repeat this process.
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<br>
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Bring the ice bucket with the cell sample(s) and FACS buffer to your bench. The following steps do not need to be carried out under sterile conditions. However, if you do not have any 1xPBS at your bench, please go to the tissue culture room and make an aliquot in a 50 mL conical in the biosafety cabinet, then keep the 50 mL (labeled "non sterile 1xPBS") at your bench. Do not handle dedicated TC reagents outside of the TC room.<br>
+
Bring the ice bucket with the cell sample(s) and FACS buffer to your bench. The following steps do not need to be carried out under sterile conditions. However, if you do not have any 1xPBS at your bench, please go to the tissue culture room and make an aliquot in a 50 mL conical in the biosafety cabinet, then keep the 50 mL (labeled "non sterile 1xPBS") at your bench. Do not handle dedicated TC reagents outside of the TC room.
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<br><br>
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'''Paraformaldehyde Fixation'''<br>
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'''Paraformaldehyde Fixation'''
# Make '''1% Paraformaldehyde Solution''': add 320 μL 32% paraformaldehyde to 680 μL 1x PBS (final volume = 1 mL for one sample)
# Make '''1% Paraformaldehyde Solution''': add 320 μL 32% paraformaldehyde to 680 μL 1x PBS (final volume = 1 mL for one sample)
# Add 1ml of '''1% Paraformaldehyde Solution''' and incubate for 15 minutes on ice. (Longer incubations compromise the quality of CV’s in DNA histograms.)
# 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 the cells 2x with cold FACS buffer: 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. Repeat this process.
# Wash the cells 2x with cold FACS buffer: 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. Repeat this process.
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<br>
'''Saponin Permeabilization'''
'''Saponin Permeabilization'''

Revision as of 11:13, 7 June 2013

<- Back to Protocols


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 experimental sample. Scale-up as needed.

Harvest the cells

  1. Obtain an ice bucket. Aliquot 4 mL FACS buffer (for one sample) into a conical tube. Chill on ice.
  2. Harvest ~2x106 cells (~1-2 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. Wash the cells 2x with cold FACS buffer: 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. Repeat this process.


Bring the ice bucket with the cell sample(s) and FACS buffer to your bench. The following steps do not need to be carried out under sterile conditions. However, if you do not have any 1xPBS at your bench, please go to the tissue culture room and make an aliquot in a 50 mL conical in the biosafety cabinet, then keep the 50 mL (labeled "non sterile 1xPBS") at your bench. Do not handle dedicated TC reagents outside of the TC room.

Paraformaldehyde Fixation

  1. Make 1% Paraformaldehyde Solution: add 320 μL 32% paraformaldehyde to 680 μL 1x PBS (final volume = 1 mL for one sample)
  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.)
  3. Wash the cells 2x with cold FACS buffer: 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. Repeat this process.


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.
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