More astrocyte and motor neuron maturation!
We are maturing another batch of astrocytes and motor neurons for a new experiment.
The experiment (2 parts):
Part 1: Astrocyte treatment
Treat astrocytes with BX559 (+/- BX559), or BX559 co-dosed with 4 different BX559 inhibitors (X, Y, Z, and W) that GenieUs identified in their in-silico screening.
After 24h, astrocyte supernatant is tested for TNFα, IL-1β, IL-8, and IL-6 to assess levels of inflammation.
Part 2: Motor neuron treatment
Healthy motor neurons will be treated with astrocyte conditioned media (+/- BX559, or BX559 co-dosed with 4 different BX559 inhibitors (X, Y, Z, and W)).
Cell viability will be monitored after 72h.
In previous experiments, we determined that incubating the astrocytes with 0.8-1.6ng/ml of BX559 for 24 hours produced optimal levels of inflammation. Therefore, we plan to replicate these conditions to induce an inflammatory response in our astrocytes.
Below are brightfield images of our astrocytes at passage 4.
Immunofluorescence imaging (IF) was performed to ensure differentiation.
In the IF images below, there are three stains:
Blue – Nuclei
Red – S100 calcium-binding protein B (S100b)
Green – N-Myc downstream-regulated gene 2 (NDRG2)
For descriptions of each of these markers, please refer to week 3s post.
Below are brightfield images of our motor neurons at day 10.
As usual, on day 10 post seeding, motor neurons are assessed for the expression of two specific neuronal markers.
1. Blue – Nuclei
2. Red – Beta III tubulin (S100b)
3. Choline acetyltransferase (NDRG2)
For descriptions of each of these markers, please refer to week 6s post.
This week was concentrated on maturing the cells. Next week, we will have the results of the 2-part experiment. Make sure you stay up to date at 12wdc.com.