Difference: ProtocolsIntrocellularROSDetection (1 vs. 5)

Revision 52017-11-09 - DaciaLeon

 
META TOPICPARENT name="ProtocolList"

Measuring Intracellular Reactive Oxygen Species (ROS)

This procedure is commonly utilized to quantify ROS. For more information about limitations associated with this assay and other methods to measure ROS, please read this helpful paper. This protocol was adapted from methods found in here.

  1. Grow 5 mL overnight cultures of your favorite strains.
  2. The following day, dilute back each culture 1:100 into fresh media.
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  1. Once the cells have reached mid-exponential phase (OD of ~0.5), pull the cultures out of the incubator and at this point you can add an ROS elicitor if desired. Some common ROS elicitors include potassium tellurite (K2TeO3), chromate (K2CrO4), and hydrogen peroxide (H2O2). Each of these compounds will induce the formation of different reactive oxygen species, so you will want to pick one (or all) that are suitable for your experiment. Additionally, it is good practice to measure ROS in the absence of any ROS elicitor.
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  1. Once the cells have reached mid-exponential phase (OD of ~0.5), you can add an ROS elicitor if desired. Some common ROS elicitors include potassium tellurite (K2TeO3), chromate (K2CrO4), and hydrogen peroxide (H2O2). Each of these compounds will induce the formation of different reactive oxygen species, so you will want to pick one (or all) that are suitable for your experiment. Additionally, it is good practice to measure ROS in the absence of any ROS elicitor.
 
  1. For ROS induction, we commonly use 0.5μg/ml of K2TeO3 and expose the cells for 30 minutes, shaking at 37°C.
  2. Centrifuge cells at 3,000g for 5 minutes at 4°C, wash once with cold saline phosphate buffer (pH = 7.3), and resuspend in 2X the initial volume with phosphate buffer.
    • Recipe for the saline phosphate buffer = 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 2mM KH2PO4, make sure to pH the buffer to 7.3 using NaOH
  3. Next, you will need to expose the cells to the ROS fluorescent dye, 2',7'-dihydrodichlorofluorescein diacetate (100mM final concentration). Perform the incubation in a 37°C water bath for 60 minutes.
  4. Centrifuge the cells at 3,000g for 5 minutes at 4°C, wash once with cold phosphate buffer, and resuspend washed cells in phosphate buffer.
  5. The fluorescence can be measured in a plate reader or by flow cytometry, excitation = 490 and emission = 519.

Revision 42017-07-31 - DaciaLeon

 
META TOPICPARENT name="ProtocolList"
Changed:
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<		There was a problem retrieving the page: Authorization Required
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Measuring Intracellular Reactive Oxygen Species (ROS)

Added:
>
>		 This procedure is commonly utilized to quantify ROS. For more information about limitations associated with this assay and other methods to measure ROS, please read this helpful paper. This protocol was adapted from methods found in here.

  1. Grow 5 mL overnight cultures of your favorite strains.
  2. The following day, dilute back each culture 1:100 into fresh media.
  3. Once the cells have reached mid-exponential phase (OD of ~0.5), pull the cultures out of the incubator and at this point you can add an ROS elicitor if desired. Some common ROS elicitors include potassium tellurite (K2TeO3), chromate (K2CrO4), and hydrogen peroxide (H2O2). Each of these compounds will induce the formation of different reactive oxygen species, so you will want to pick one (or all) that are suitable for your experiment. Additionally, it is good practice to measure ROS in the absence of any ROS elicitor.
  4. For ROS induction, we commonly use 0.5μg/ml of K2TeO3 and expose the cells for 30 minutes, shaking at 37°C.
  5. Centrifuge cells at 3,000g for 5 minutes at 4°C, wash once with cold saline phosphate buffer (pH = 7.3), and resuspend in 2X the initial volume with phosphate buffer.
    • Recipe for the saline phosphate buffer = 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 2mM KH2PO4, make sure to pH the buffer to 7.3 using NaOH
  6. Next, you will need to expose the cells to the ROS fluorescent dye, 2',7'-dihydrodichlorofluorescein diacetate (100mM final concentration). Perform the incubation in a 37°C water bath for 60 minutes.
  7. Centrifuge the cells at 3,000g for 5 minutes at 4°C, wash once with cold phosphate buffer, and resuspend washed cells in phosphate buffer.
  8. The fluorescence can be measured in a plate reader or by flow cytometry, excitation = 490 and emission = 519.
 

Revision 32017-07-30 - DaciaLeon

 
META TOPICPARENT name="ProtocolList"
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Measuring intracellular ROS:

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>		There was a problem retrieving the page: Authorization Required
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Protocol:

1.) Grow 5mL overnight cultures of the strains to be tested.

2.) The following day, dilute back the cultures 1:100 into fresh media.

3.) Grow the cells to mid-exponential phase (OD = ~0.5). At this point, you may want to add an ROS elicitor to your culture (ex, H2O2, K2TeO3,

4.) Stress ce

Overnight culture was passaged 1 : 100 into fresh medium.

Monitor the obsorbance to 0.2-0.4.

Exposed to K2TeO3 (0.5 µg/ml) for 30 min.

Centrifuge at 3,000 g for 5 min at 4°C , cells were washed with ice chilled saline phosphate buffer (137 mM NaCl 2.7 mM KCl, 10 mM Na2HPO4 , 2mM KH2PO4, pH 7.3) and resuspended with two volume of the same buffer, conducted at 4°C .

Cells were incubated with 100 uM (50 µg/ml) 2’,7’-dihydrodichlorofluorescein diacetate at 37°C for 60 min in water bath.

Centrifuge at 3,000 g for 5 min at 4°C. Wash once with ice chilled PBS. Centrifuge and put on ice before run flow.

Fluorescence intensity was determined by flow cytometry.

-- Main.XueZhang - 25 May 2017

 

Revision 22017-07-29 - DaciaLeon

 
META TOPICPARENT name="ProtocolList"

Measuring intracellular ROS:

Protocol:

Added:
>
>		 1.) Grow 5mL overnight cultures of the strains to be tested.

2.) The following day, dilute back the cultures 1:100 into fresh media.

3.) Grow the cells to mid-exponential phase (OD = ~0.5). At this point, you may want to add an ROS elicitor to your culture (ex, H2O2, K2TeO3,

4.) Stress ce

  Overnight culture was passaged 1 : 100 into fresh medium.

Monitor the obsorbance to 0.2-0.4.

Exposed to K2TeO3 (0.5 µg/ml) for 30 min.

Centrifuge at 3,000 g for 5 min at 4°C , cells were washed with ice chilled saline phosphate buffer (137 mM NaCl 2.7 mM KCl, 10 mM Na2HPO4 , 2mM KH2PO4, pH 7.3) and resuspended with two volume of the same buffer, conducted at 4°C .

Cells were incubated with 100 uM (50 µg/ml) 2’,7’-dihydrodichlorofluorescein diacetate at 37°C for 60 min in water bath.

Centrifuge at 3,000 g for 5 min at 4°C. Wash once with ice chilled PBS. Centrifuge and put on ice before run flow.

Fluorescence intensity was determined by flow cytometry.

-- Main.XueZhang - 25 May 2017

Revision 12017-05-25 - XueZhang

 
META TOPICPARENT name="ProtocolList"

Measuring intracellular ROS:

Protocol:

Overnight culture was passaged 1 : 100 into fresh medium.

Monitor the obsorbance to 0.2-0.4.

Exposed to K2TeO3 (0.5 µg/ml) for 30 min.

Centrifuge at 3,000 g for 5 min at 4°C , cells were washed with ice chilled saline phosphate buffer (137 mM NaCl 2.7 mM KCl, 10 mM Na2HPO4 , 2mM KH2PO4, pH 7.3) and resuspended with two volume of the same buffer, conducted at 4°C .

Cells were incubated with 100 uM (50 µg/ml) 2’,7’-dihydrodichlorofluorescein diacetate at 37°C for 60 min in water bath.

Centrifuge at 3,000 g for 5 min at 4°C. Wash once with ice chilled PBS. Centrifuge and put on ice before run flow.

Fluorescence intensity was determined by flow cytometry.

-- Main.XueZhang - 25 May 2017

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