Haynes:ICCSaponin

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'''Intracellular Protein Staining with Saponin Permeabilization'''<br>
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=Intracellular Protein Staining with Saponin Permeabilization=
Karmella Haynes 2013<br>
Karmella Haynes 2013<br>
Adapted from protocol by Jodene K. Moore, Harvard Systems Biology, 2013<br>
Adapted from protocol by Jodene K. Moore, Harvard Systems Biology, 2013<br>
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Reagents:
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REAGENTS:
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* '''Paraformaldehyde''' (EMS Cat # 15714) - 32% Solution, EM Grade, MeOH-free  
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* '''32% 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)
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* '''FB-S''' Saponin Staining/Wash Buffer (Sigma Catalog # S4521) - 10% stock (stored at 4°C)  
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* '''10% 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.
 +
* Primary antibody
 +
* Secondary antibody (if primary is not dye-conjugated)
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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 two samples. One stained experimental sample and one unstained flow cytometry "blank" control. Scale-up as needed.''
 +
<br>
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'''Harvest the cells'''
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'''Harvest the cells''' - do the following in the TC room
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# Obtain an ice bucket. Aliquot 4 mL '''FACS buffer''' (for one sample) into a conical tube. Chill on ice.
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# Obtain an ice bucket. Aliquot 5 mL '''FACS buffer''' per sample (10 mL) into a conical tube. Chill on ice.
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# Harvest ~2x10<sup>6</sup> cells (~1-2 wells of adherent mammalian cells from a 6-well plate) using the standard trypsinization procedure.
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# Harvest ~2x10<sup>6</sup> cells (~1-2 wells of adherent mammalian cells from a 6-well plate) per sample using the standard trypsinization procedure.
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# Collect the cells in growth medium and transfer to a 15 mL conical tube.
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# Collect the cells in growth medium and transfer to 15 mL conical tubes.
# Pellet the cells at room temperature at 1000 rpm for 3 min.
# Pellet the cells at room temperature at 1000 rpm for 3 min.
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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.
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# Aspirate off the growth medium, but leave enough to cover the pellets. Flick each tube to break up the pellets. Chill the cells on ice.
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# 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 each sample. Pellet the cells at room temperature at 1000 rpm for 3 min. Aspirate off the buffer, leaving enough to cover the pellets. Flick each tube to break up the pellets. Repeat this process.
 +
<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 samples and cold 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><br>
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'''Paraformaldehyde Fixation'''<br>
+
'''Paraformaldehyde Fixation''' - keep the cells on ice as much as possible
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# Make '''1% Paraformaldehyde Solution''': add 320 μL 32% paraformaldehyde to 680 μL 1x PBS (final volume = 1 mL for one sample)
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# Make fresh '''1% Paraformaldehyde Solution''': add 62.5 μL 32% paraformaldehyde to 1937.5 μL 1x PBS (final volume = 2 mL; 1 mL for each sample)
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# Add 1ml of '''1% Paraformaldehyde Solution''' and incubate for 15 minutes on ice. (Longer incubations compromise the quality of CV’s in DNA histograms.)
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# Label empty 1.5 mL microfuge tubes (one per sample).
-
# 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.
+
# Add 1ml of '''1% Paraformaldehyde Solution''' to the cells in each 15 mL conical, transfer cells to the appropriate labeled 1.5 mL tube, and incubate for '''12 min. on ice'''. (Longer incubations compromise the quality of CV’s in DNA histograms.)
 +
# Immediately pellet the cells at room temperature at 1000 rpm for 3 min. Discard the buffer into a conical or beaker (so that you an later discard the paraformaldehyde as has waste). Flick each tube to break up the pellets.
 +
# 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. Discard the buffer. Flick each tube to break up the pellets. Repeat this process.
 +
<br>
'''Saponin Permeabilization'''
'''Saponin Permeabilization'''
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• Decant the last wash and resuspend pellet in remaining residual volume.
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# Make fresh '''0.2% FB-S''' for the cells: add 120 μL 10% FB-S to 5880 μL cold FACS buffer (final volume = 6 mL; 3 mL for each sample)
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Resuspend cells in 1 ml FB-S, added slowly while flicking the sample.
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# Resuspend cells in 1 ml '''0.2% FB-S''', added slowly while flicking the sample.
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Incubate at RT for 30 minutes.
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# Incubate at room temperature for 30 minutes.
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• Spin down. Decant. Resuspend cell in the residual volume of FB-S. Should be about 100ul.
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# Pellet the cells at room temperature at 1000 rpm for 3 min. Pipette-off the supernatant, leaving a ~100 μL volume of cells plus buffer in the tube. Flick each tube to break up the pellets.
 +
<br>
 +
'''Antibody Staining''' - All staining and washes are to be done in '''0.2% FB-S'''. Do not stain the "blank" control cells. If the Primary antibody is dye-conjugated try to keep the samples protected from light as much as possible.
 +
# Make fresh '''0.2% FB-S''' for the antibody dilutions: add 20 μL 10% FB-S to 980 μL cold FACS buffer (final volume = 1 mL)
 +
# Dilute '''Primary antibody''' in 0.2% FB-S: make 10 μL diluted antibody that is 10X more concentrated than the manufacturer's/ published protocol's recommendation. This is enough for 10 experimental samples.
 +
# Add 1 μL of '''diluted Primary antibody''' <u>to the 100 μL experimental sample ONLY</u>. Incubate for 30 min. on ice.
 +
# Wash the cells 2x with cold 0.2% FB-S buffer: Add 1 mL '''cold 0.2% FB-S buffer''' to the cells. Pellet the cells at room temperature at 1000 rpm for 3 min. Discard the buffer. Flick each tube to break up the pellets. Repeat this process.
 +
# Secondary antibody: If the primary is dye-cojugated, proceed to '''Flow Cytometry'''. If the primary is not dye-conjugated, repeat the steps above for the dye-conjugated secondary. Try to keep the samples protected from light as much as possible.
 +
<br>
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Antibody Staining (All staining and washes are to be done in FB-S)
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'''Flow cytometry'''
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• Incubate cells in 100ul final volume of antibody(ies), diluted to recommended concentration in FB-S, for 30 minutes on ice.
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# Resuspend each cell sample in 500ul of '''cold FACS Buffer'''.
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• Wash cells 2X in cold FB-S. Decant Supernatant.  
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# Using a 1000 μL micropipette, force each 500 μL of cells through a separate, clean FACS tube strainer cap.
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• Resuspend in 500ul of FACS Buffer.
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# Analyze on Flow cytometer. (keep covered on ice until analysis).
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Analyze on Flow cytometer. (keep covered on ice until analysis; always have NEGATIVE control)  
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# Dilute the sample with additional cold FACS buffer if the reads per second exceed 250 (Wang lab's bench top flow cytometer).
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2nd Antibody Staining
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• Follow directions as above in Antibody Staining.
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Current revision

<- Back to Protocols


Intracellular Protein Staining with Saponin Permeabilization

Karmella Haynes 2013
Adapted from protocol by Jodene K. Moore, Harvard Systems Biology, 2013


REAGENTS:

  • 32% 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)
  • 10% 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.
  • Primary antibody
  • Secondary antibody (if primary is not dye-conjugated)


PROTOCOL
This procedure is for two samples. One stained experimental sample and one unstained flow cytometry "blank" control. Scale-up as needed.

Harvest the cells - do the following in the TC room

  1. Obtain an ice bucket. Aliquot 5 mL FACS buffer per sample (10 mL) into a conical tube. Chill on ice.
  2. Harvest ~2x106 cells (~1-2 wells of adherent mammalian cells from a 6-well plate) per sample using the standard trypsinization procedure.
  3. Collect the cells in growth medium and transfer to 15 mL conical tubes.
  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 pellets. Flick each tube to break up the pellets. Chill the cells on ice.
  6. Wash the cells 2x with cold FACS buffer: Add 1 mL cold FACS buffer to each sample. Pellet the cells at room temperature at 1000 rpm for 3 min. Aspirate off the buffer, leaving enough to cover the pellets. Flick each tube to break up the pellets. Repeat this process.


Bring the ice bucket with the cell samples and cold 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 - keep the cells on ice as much as possible

  1. Make fresh 1% Paraformaldehyde Solution: add 62.5 μL 32% paraformaldehyde to 1937.5 μL 1x PBS (final volume = 2 mL; 1 mL for each sample)
  2. Label empty 1.5 mL microfuge tubes (one per sample).
  3. Add 1ml of 1% Paraformaldehyde Solution to the cells in each 15 mL conical, transfer cells to the appropriate labeled 1.5 mL tube, and incubate for 12 min. on ice. (Longer incubations compromise the quality of CV’s in DNA histograms.)
  4. Immediately pellet the cells at room temperature at 1000 rpm for 3 min. Discard the buffer into a conical or beaker (so that you an later discard the paraformaldehyde as has waste). Flick each tube to break up the pellets.
  5. 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. Discard the buffer. Flick each tube to break up the pellets. Repeat this process.


Saponin Permeabilization

  1. Make fresh 0.2% FB-S for the cells: add 120 μL 10% FB-S to 5880 μL cold FACS buffer (final volume = 6 mL; 3 mL for each sample)
  2. Resuspend cells in 1 ml 0.2% FB-S, added slowly while flicking the sample.
  3. Incubate at room temperature for 30 minutes.
  4. Pellet the cells at room temperature at 1000 rpm for 3 min. Pipette-off the supernatant, leaving a ~100 μL volume of cells plus buffer in the tube. Flick each tube to break up the pellets.


Antibody Staining - All staining and washes are to be done in 0.2% FB-S. Do not stain the "blank" control cells. If the Primary antibody is dye-conjugated try to keep the samples protected from light as much as possible.

  1. Make fresh 0.2% FB-S for the antibody dilutions: add 20 μL 10% FB-S to 980 μL cold FACS buffer (final volume = 1 mL)
  2. Dilute Primary antibody in 0.2% FB-S: make 10 μL diluted antibody that is 10X more concentrated than the manufacturer's/ published protocol's recommendation. This is enough for 10 experimental samples.
  3. Add 1 μL of diluted Primary antibody to the 100 μL experimental sample ONLY. Incubate for 30 min. on ice.
  4. Wash the cells 2x with cold 0.2% FB-S buffer: Add 1 mL cold 0.2% FB-S buffer to the cells. Pellet the cells at room temperature at 1000 rpm for 3 min. Discard the buffer. Flick each tube to break up the pellets. Repeat this process.
  5. Secondary antibody: If the primary is dye-cojugated, proceed to Flow Cytometry. If the primary is not dye-conjugated, repeat the steps above for the dye-conjugated secondary. Try to keep the samples protected from light as much as possible.


Flow cytometry

  1. Resuspend each cell sample in 500ul of cold FACS Buffer.
  2. Using a 1000 μL micropipette, force each 500 μL of cells through a separate, clean FACS tube strainer cap.
  3. Analyze on Flow cytometer. (keep covered on ice until analysis).
  4. Dilute the sample with additional cold FACS buffer if the reads per second exceed 250 (Wang lab's bench top flow cytometer).
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