Neutrophil transmigration through Parylene membranes
Tom came up with this idea of using these membrane with precise thickness and pore size control to obtain a critical thickness and pore size, beyond which neutrophils cannot go transmigrate. N-Formylmethionyl-leucyl-phenylalanine (fMLP) was used in the lower channel to serve as a chemotactic factor for transmigration. Additionally, for membranes with pore sizes or thicknesses close to the critical values, the behavior of neutrophils can be explored to determine what consequences passing through these confinements might have for the neutrophils.
Here is the basic preliminary design by Alec:
The protocol can be found here:
https://drive.google.com/open?id=1qKYOY46Lcnu6qYncw7wllikbhCgdP7YM
Here is the first set of experiments with two thickness:
700nm (1um pore size):
https://drive.google.com/open?id=1hPsVZJ9wHWyR5pkGKQmc9BjeOP17te7r
1200nm (1um pore size)
https://drive.google.com/open?id=1pOvjXQ-uSZ1n7YH3aAWXlJPa-J6BpAP0
Needless to say, they were total failures. We had trouble wetting the lower channel.
Then, I improved the design of device using help from Zahra and Henry, changing the channel size and the layers. In this set of experiments, we explored whether neutrophils simply do not like transmigration through Parylene. For this purpose, we started the experiments with using 3 um pore sizes:
https://drive.google.com/open?id=1eVkPMQcADKqYYYvIR2_-5456BZ8m8kAj
Then, we continued with 1 um pore size:
https://drive.google.com/open?id=1_IVc7zO2zuuI1mNnQDUGahBmfGa5z9C_
As you see, there was transmigration both through 1um and 3um pores. These initial experiments showed the feasibility of these study. For future experiments, we still need improvement for the devices, the main problem being that once you fill the chambers, there is fluid exchange between the chamber, with fMLP going to the top channel, compromising the experiment. Also, more effective surface can be used.
Bonus video:
https://drive.google.com/open?id=1ocjR6NaMFavrDZyM-ill78EUmAQTEVb3