Choosing the Most Relevant Normalization Method

For some time I have been trying to work out how to normalise my OXPHOS sample prior to doing an assay. Finally found an Agilent paper "Methods and strategies for normalizing XF metabolic data to cellular parameters" which shows the following schematic solving one of my problems ie if I do not have access to a Cytation unit to automatically stain, count and photograph the cells, what method should I use?

Methods and strategies for normalizing XF metabolic data to cellular parameters

It would seem that unless we can find a Cytation to use, teh methods I can use to normalise my assay data are :

1.  Protein measurement,
2.  gDNA measurement, or
3.  Manual counting after cell dissociation

Locating healthy control blood

There are a few a few problems that I face with respect to the sourcing, collection, preparation and storage of the blood samples, namely:

1. We can get the Parkinson's blood from Sunshine hospital neurology clinic, but we are yet to work out where we can get age matched blood from healthy controls. One idea is to ask their partners, 
2. Once we have the blood, what are the laboratory protocols we should use for optimally preparing the blood ie centrifugation. Too slow and the whole blood does not seperate into the buffy coat, whilst too fast will damage the cells, and,
3. Once we have isolated the leukocytes, what is the best way and what temperature should we store the remaining blood samples and the prepared leukocytes.

I made contact with the Melbourne Blood Bank today and had a series of phone conversations which I will summarise below:

• The blood bank can provide us with a sample of blood for us to test and work out the best parameters to prepare, isolate the buffy coat and then store the remaining blood and isolated leukocytes – the cost of this sample blood is free to authorised research projects
• If you are an authorised project you need a permit to receive the blood. In a conversation with Francesca Hulme (Senior Transfusion Scientist), she is sending my details to Andrew Morrissey who is a senior scientist in the blood banks research supply chain, as she thinks he can provide me with the details around how we might qualify for research blood samples for free and be able to send us the paper work to get the process of approval and a permit organised.
• The blood bank research group have a project running under the leadership of Dr Lacey Johnson (Principal Research Fellow in the Product Development Research Group) looking at methods to extend the shelf life of blood products. Francesca will speak to Dr Lacey about our blood needs with respect to how we should store our whole blood and our isolated buffy coat and leukocytes.
• I also spoke with Francesca about preparation methods i.e. centrifugation, and she said that she spins down whole blood to get the buffy coat and then they filter out the leukocytes to leave just the platelets, where as we want to filter out the platelets. I asked if we could get a copy of their protocol and she was going to see if this could be sent to us. Francesca also said that Michael Wheeler at Monash medical centre had done a bit of work in separating out leukocytes and he may be willing to talk to me about the best way to isolate the leukocytes. She said she would pass my details onto Michael and ask him to ring me.
• Finally, since the blood bank can provide us a healthy blood sample, I explained to Francesca that whilst we had made initial arrangements to acquire some Parkinson’s blood, we were still thinking about how we could get age matched healthy blood for the Parkinson’s age groups and the young 30-40 yrs age group for the biomarker study. Francesca said that if we are eligible for the sample and get a permit they can also supply us with sufficient blood samples to match all our healthy controls for free.

I have my fingers crossed that we will be seen as eligible to get the blood and for free.

XF Real-Time ATP Rate Assay well plate set up

Cellular metabolic regulation allows cells to adjust for changes in ATP demand with subsequent changes in ATP production to maintain total intracellular ATP levels. By using the Agilent Seahorse XF Real-Time ATP Rate Assay we can measure total ATP production rates in living cells and to distinguish between the fractions of ATP that are produced from mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis, the two main metabolic pathways responsible for ATP production in mammalian cells. 

The XF Real-Time ATP Rate Assay can also be used to calculate the ideal cell seeding density. By creating four different seeding density sources ie 0.5X, 1.0X, 2.0X and 4.0X we can determine which level for our particular cells is best. Optimal cell seeding numbers vary widely, though are typically between 5 × 10³ – 4 × 10⁴ cells per well and must be determined empirically. (For example, for 1 x 10⁴ cells per well, resuspend cells 1 x 10⁴ per 80 μL = 1.25 x 10⁵ cells per mL)

To simultaneously test the ideal seeding level for both Parkinson's and healthy control cells the following well plate design is proposed.