TITLE:  Primate Hair Cortisol Processing l
AUTHORS: Kimberley Phillips, Matthew Lopez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a protocol to quantify cortisol concentration from hair. Cortisol is a glucocorticoid hormone which is prominent in primates and can be used as a retrospective biomarker of hypothalamic-pituitary-adrenal (HPA) axis activity. </div><br/><div class = "text-block">Our method is adapted from a protocol used in the Meyer Lab (Meyer et al. 2014). </div><br/><br/><div class = "text-block">Reference:</div><br/><div class = "text-block"><span>Meyer, J., Novak, M., Hamel, A., & Rosenberg, K. (2014). Extraction and analysis of cortisol from human and monkey hair.</span><span style = "font-style:italic;">Journal of visualized experiments : JoVE</span><span>, (83), e50882. https://doi.org/10.3791/50882</span></div></div>

[STEPS]
?. [Preparation before obtaining shaved hair ]
Hair clippers should be cleaned with alcohol before and after each use.
?. [Obtaining shaved hair ]
Shave the hair from the upper back of the animal using electric clippers.
?. [Obtaining shaved hair ]
The hair should be shaved close without nicking the skin, as this can cause falsely elevated concentration of cortisol due to blood being present in the sample. It is essential that hair be collected from the same body region across all animals for a given study.
?. [Obtaining shaved hair ]
The distal end of each sample is then wrapped in masking tape, leaving 1–1.5 cm of the proximal hair exposed. The sample can be placed in an envelope and stored at -20 °C (-4 °F) or room temperature out of direct sunlight until further processing.
?. [Obtaining shaved hair ]
Envelopes should be labelled with species name, subject ID, sex, DOB (date of birth), DOC (date of collection), location of hair sample, and project affiliation code.
?. [Preparation of weigh and cutting samples]
Calibrate the analytical scale.
?. [Preparation of weigh and cutting samples]
Clean instruments (forceps, spatula) with isopropyl alcohol wipes before and after each use.
?. [Preparation of weigh and cutting samples]
Cut a 7cm x 7 cm square from printer paper for each hair sample.
?. [Preparation of weigh and cutting samples]
Create 4–5 labels for each sample with the information supplied in step 4.
?. [Weighing and cutting samples]
Weigh the 7cm x 7cm paper on the analytical scale and record the weight.
?. [Weighing and cutting samples]
Tare the piece of paper before adding the hair.
?. [Weighing and cutting samples]
Carefully remove the hair sample from the container while wearing laboratory gloves. Identify the shaved end of the sample.
?. [Weighing and cutting samples]
Using scissors and a ruler cut 1-1.5cm of hair from the exposed hair. Depending upon the rate of hair growth for the specific species and individual, this will reflect hair cortisol from the most recent 1–1.5 months.
?. [Weighing and cutting samples]
Transfer the hair on the square paper and weigh the cut sample. Record the weight.
?. [Weighing and cutting samples]
Use forceps to grab the hair and store it in the polypropylene tube.
?. [Weighing and cutting samples]
Place the label for that subject on the polypropylene tube.
?. [Washing the hair]
Use the motorized pipette controller and 10 mL serological pipette to add 5 mL of HPLC grade isopropanol to each 15 mL polypropylene tube.
?. [Washing the hair]
Invert the sample for 3 minutes on the rotator, using speed 12.
?. [Washing the hair]
Use a glass funnel to decant the isopropanol into a waste container.
?. [Washing the hair]
Carefully decant the isopropanol and use a spatula to catch any hair that maybe traveling down the tube as you decant.
?. [Washing the hair]
Repeat steps 16–18 twice.
?. [Washing the hair]
Set the tubes on a rack in the fume hood with the lids off allowing the hair to completely dry. This will take 4 - 5 days.
?. [Grinding the hair]
Separate each washed and dried hair sample in half and place each half into a mill jar. Grind the hair to a fine powder using the ball mill, for 6 minutes at 25 Hz.
?. [Grinding the hair]
With the scapula scrape the sides of the jar to get all powdered hair free from the walls. Collect the hair and put it in the appropriate labeled microcentrifuge tube and weigh it.
?. [Preparation for grinding the hair]
Place the sample ID label (step 8) on a  2 mL microcentrifuge tube.
?. [Preparation for grinding the hair]
Weigh the microcentrifuge tube (with the label affixed) , then tare.
?. [Weighing and cutting samples]
Clean the forceps with isopropanol before using it again for the next hair sample.
?. [Grinding the hair]
Once the appropriate amount has been placed in the microcentrifuge tube, store any remaining powdered hair in another labelled 2 mL microcentrifuge tube and place it in the refrigerator or at room temperature.
?. [Grinding the hair]
The weight of powdered hair in the microcentrifuge tube needed for the species is indicated below: Capuchins, marmosets and tamarins, 50mg (0.05g) Chimpanzees, 40mg (0.04g) It is essential to keep careful records of the subject ID and the weight of each sample.
?. [ Methanol Extraction]
Use the motorized pipette controller and a serological pipette to add 1 mL HPLC grade methanol to each microcentrifuge tube.
?. [ Methanol Extraction]
Place the tube on a multi-purpose tube rotator for 24 hours. Set the time for 24:00 (24 hours) and run at speed 12.
?. [ Methanol Extraction]
Plan accordingly to be available to extract the methanol at the conclusion of the 24 hours.
?. [ Methanol Extraction]
At the end of the 24 hours the samples must immediately be centrifuged at 14,000 RPM for 3 minutes.
?. [ Methanol Extraction]
Collect 0.6 mL (600 microliters) of supernatant into a 1.5 mL microcentrifuge tube. Do not disturb the pellet of hair.
?. [Drying down the methanol]
Steps 30–37 will take approximately 45 minutes - 1 hour.Using the MicroVap underneath the fume hood turn on the nitrogen tank.
?. [Drying down the methanol]
Place each sample onto the bed noting where each sample is placed.
?. [Drying down the methanol]
Position the nitrogen apparatus over the top of the samples so the tips will be in the center of each microcentrifuge tube.
?. [Drying down the methanol]
Carefully lower until stop and lock into place.
?. [Drying down the methanol]
Turn the unit on.Make small turns on the nitrogen gas knob on the side of the panel of the hood, also adjust the gauge of the MicroVap to be sure it is open.
?. [Drying down the methanol]
Slowly increase the amount of nitrogen being released using the knob on the hood until you see the gauge at approximately 10.
?. [Drying down the methanol]
Turning on the gas too fast will cause the methanol in each sample to splash out.Going slower is better in order to prevent any samples from splashing methanol.
?. [Drying down the methanol]
Once the nitrogen steam is at the desired intensity, wait for the evaporation to occur and keep monitoring the sample. Increase the flow of nitrogen as needed.
?. [Drying down the methanol]
The evaporation takes about 45 to 1 hour depending on the intensity of the nitrogen steam.After use clean the metal pins with alcohol.
?. [Preforming cortisol assay]
Allow samples to thaw before diluting for the assay.
?. [Preforming cortisol assay]
Dilute the sample.Capuchins, marmosets, and tamarins, dilution is 1:40Chimpanzees, no dilution is necessary
?. [Preforming cortisol assay]
Follow the procedure in the Salimetrics Manual for Salivary Cortisol.
?. [Weighing and cutting samples]
Repeat steps 9–15 for each sample.
?. [Drying down the methanol]
Reconstitute each sample with PBS.
?. [Drying down the methanol]
For capuchins, marmosets and tamarins, use 200 micrometers of PBS buffer.For chimpanzees, use 100 micrometers of PBS buffer.
?. [Drying down the methanol]
Cover the top of each microcentrifuge tube with parafilm and store at -20 C until ready to perform assay.