Difference between revisions of "UW-Stout/Hydroxychloroquine SP21"

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* [https://www.corning.com/worldwide/en/products/life-sciences/keymatch/3370.html Corning COSTAR 96-well clear flat-bottom assay plate]
 
* [https://www.corning.com/worldwide/en/products/life-sciences/keymatch/3370.html Corning COSTAR 96-well clear flat-bottom assay plate]
 
* Wild-type yeast in 2x synthetic complete media at an OD600 of 0.1-0.2
 
* Wild-type yeast in 2x synthetic complete media at an OD600 of 0.1-0.2
−
* Hydroxychloroquine, 300 ug/uL solution in sterile water
+
* Hydroxychloroquine, 300 ug/uL solution in sterile water (as determined by the calibration protocol)
 
* P20, P200 micropipettors
 
* P20, P200 micropipettors
  
 
==Equipment==
 
==Equipment==
 
* [https://www.moleculardevices.com/sites/default/files/en/assets/data-sheets/br/spectramax-plus-384-microplate-reader.pdf Molecular Devices SpectraMax Plus 384 Microplate Reader]
 
* [https://www.moleculardevices.com/sites/default/files/en/assets/data-sheets/br/spectramax-plus-384-microplate-reader.pdf Molecular Devices SpectraMax Plus 384 Microplate Reader]
 +
==Precautions==
 +
Use gloves and safety glasses for this experiment. Hydroxychloroquine is a potent chemical used to treat malaria and some autoimmune conditions. However, side effects include but are not limited to nausea, headache, dizziness, rash.
 +
==Calibration Protocol==
 +
#Vortex the yeast culture briefly to resuspend the yeast cells.
 +
#Prepare a stock solution of hydroxychloroquine 3000 ug/mL.
 +
#Set up 8 wells as follows:
 +
#*Well 1: 3.3uL HCQ, 46.7uL sterile water (final concentration 100ug/mL)
 +
#*Well 2: 10uL HCQ, 40uL sterile water (final concentration 300ug/mL)
 +
#*Well 3: 16.7uL HCQ, 33.3uL sterile water (final concentration 500ug/mL)
 +
#*Well 4: 23.3uL HCQ, 26.7uL sterile water (final concentration 700ug/mL)
 +
#*Well 5: 30uL HCQ, 20uL sterile water (final concentration 900ug/mL)
 +
#*Well 6: 36.7uL HCQ, 13.3uL sterile water (final concentration 1100ug/mL)
 +
#*Well 7: 43.3uL HCQ, 6.7uL sterile water (final concentration 1300ug/mL)
 +
#*Well 8: 50uL HCQ (final concentration 1500ug/mL)
 +
#Add 50uL of yeast culture to each well.
 +
#Aliquot 50 uL of each stock and add 50 uL of yeast culture to each sample.
 +
#To measure cell growth over a period of 24 hours, set up the plate reader as follows:
 +
#*Temperature: 30°C
 +
#*Mode: Kinetic
 +
#*Wavelength: 600 nm
 +
#*Interval: 5 minutes
 +
#*Total run time: 24 hours
 +
#*Shake before reading: 30 seconds.
 +
#Transfer the assay plate to the reader and read for 24 hours.
 +
 +
==Raw Data from the Calibration Protocol==
 +
[[Image:Calibration.png]]
 +
 +
Concentrations in the legend are final concentrations of hydroxychloroquine.
 +
From this set of curves, a concentration of 300 ug/mL is the optimal concentration of the stressor to test the effect of hydroxychloroquine on yeast cells because it exerts enough stress over the yeast to measurably inhibit its growth, but does not prevent it from growing altogether.
  
 
==Protocol==
 
==Protocol==
−
1. Vortex the yeast culture briefly to resuspend the yeast cells.
+
Use the data from the calibration protocol for the optimal concentration of hydroxychloroquine.
−
2. Set up eight wells according to the following table:
+
# Vortex the yeast culture briefly to resuspend the yeast cells.
−
+
# Set up seven wells aliquoting 50uL of the stock hydroxychloroquine and 50uL of yeast culture. Use the seventh well for the wild-type yeast strain.
−
[[File:Capture.png]]
+
#*The final concentration of hydroxychloroquine is 300ug/mL.  
−
 
+
#To measure cell growth over a period of 24 hours, set up the plate reader as follows:
−
The final concentration of hydroxychloroquine is 300ug/mL. We have controls for this experiment: a well with the wild-type strain yeast untreated with the hydroxychloroquine solution, and wells with the modified strains yeast untreated with the hydroxychloroquine solution for all 8 modified strains. We also have experimental samples: a well with the wild-type strain yeast treated with the hydroxychloroquine solution, and wells with the modified strains yeast treated with the hydroxychloroquine solution for all 8 modified strains.
+
#*Temperature: 30°C
−
3. To measure cell growth over a period of 24 hours, set up the plate reader as follows:
+
#*Mode: Kinetic
−
Temperature: 30°C
+
#*Wavelength: 600 nm
−
Mode: Kinetic
+
#*Interval: 5 minutes
−
Wavelength: 600 nm
+
#*Total run time: 24 hours
−
Interval: 5 minutes
+
#*Shake before reading: 30 seconds
−
Total run time: 24 hours
+
#Transfer the assay plate to the reader and read for 24 hours.
−
Shake before reading: 30 seconds
+
#*Note: Repeat the experiment a few times to account for natural variation.
−
4. Transfer the assay plate to the reader and read for 24 hours.
 
−
Note: Repeat the experiment a few times to account for natural variation.
 

Latest revision as of 07:34, 28 April 2021

Materials

Equipment

Precautions

Use gloves and safety glasses for this experiment. Hydroxychloroquine is a potent chemical used to treat malaria and some autoimmune conditions. However, side effects include but are not limited to nausea, headache, dizziness, rash.

Calibration Protocol

  1. Vortex the yeast culture briefly to resuspend the yeast cells.
  2. Prepare a stock solution of hydroxychloroquine 3000 ug/mL.
  3. Set up 8 wells as follows:
    • Well 1: 3.3uL HCQ, 46.7uL sterile water (final concentration 100ug/mL)
    • Well 2: 10uL HCQ, 40uL sterile water (final concentration 300ug/mL)
    • Well 3: 16.7uL HCQ, 33.3uL sterile water (final concentration 500ug/mL)
    • Well 4: 23.3uL HCQ, 26.7uL sterile water (final concentration 700ug/mL)
    • Well 5: 30uL HCQ, 20uL sterile water (final concentration 900ug/mL)
    • Well 6: 36.7uL HCQ, 13.3uL sterile water (final concentration 1100ug/mL)
    • Well 7: 43.3uL HCQ, 6.7uL sterile water (final concentration 1300ug/mL)
    • Well 8: 50uL HCQ (final concentration 1500ug/mL)
  4. Add 50uL of yeast culture to each well.
  5. Aliquot 50 uL of each stock and add 50 uL of yeast culture to each sample.
  6. To measure cell growth over a period of 24 hours, set up the plate reader as follows:
    • Temperature: 30°C
    • Mode: Kinetic
    • Wavelength: 600 nm
    • Interval: 5 minutes
    • Total run time: 24 hours
    • Shake before reading: 30 seconds.
  7. Transfer the assay plate to the reader and read for 24 hours.

Raw Data from the Calibration Protocol

Calibration.png

Concentrations in the legend are final concentrations of hydroxychloroquine. From this set of curves, a concentration of 300 ug/mL is the optimal concentration of the stressor to test the effect of hydroxychloroquine on yeast cells because it exerts enough stress over the yeast to measurably inhibit its growth, but does not prevent it from growing altogether.

Protocol

Use the data from the calibration protocol for the optimal concentration of hydroxychloroquine.

  1. Vortex the yeast culture briefly to resuspend the yeast cells.
  2. Set up seven wells aliquoting 50uL of the stock hydroxychloroquine and 50uL of yeast culture. Use the seventh well for the wild-type yeast strain.
    • The final concentration of hydroxychloroquine is 300ug/mL.
  3. To measure cell growth over a period of 24 hours, set up the plate reader as follows:
    • Temperature: 30°C
    • Mode: Kinetic
    • Wavelength: 600 nm
    • Interval: 5 minutes
    • Total run time: 24 hours
    • Shake before reading: 30 seconds
  4. Transfer the assay plate to the reader and read for 24 hours.
    • Note: Repeat the experiment a few times to account for natural variation.
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