Preparation and Manipulation of Olfactory Epithelium Explant Cultures for Measurement of the Mechanical Tension of Individual Axons Using the Biomembrane Force Probe

[Abstract] In this paper, we describe a protocol allowing measurement of the mechanical tension of individual axons grown ex vivo from neural tissue explants. This protocol was developed with primary cultures of olfactory epithelium explants from embryonic (E13.5) mice. It includes a detailed description of explant dissection and culture, as well as the main steps of the procedure for axon tension measurement using the previously established Biomembrane Force Probe.

Note: The next steps can be done during this coating.

5.
Prepare the L-15; 5% FBS (Solution A) and store it on ice.
6. Prepare the culture medium (Solution B) and store it on ice until the explants are ready. 7. Set up the material under the hood (stereomicroscope, glass dish containing cold L-15, forceps, scalpels, tips and pipettes ( Figures 1B and 1C).

Embryo collection
Note: All animal procedures must be approved by an Ethics Committee (for our own experiments, the Ile-de-France Ethics Committee).
For each experiment, we typically prepare 20 to 30 explants from a total of 3 to 4 embryos Copyright Fouquet and Trembleau. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0). 6 www.bio-protocol.org/e3213 Bio-protocol 9(08): e3213. DOI:10.21769/BioProtoc.3213 extracted from a pregnant female at embryonic day 13.5 (e.g., 5-6 explants per dish, around 20 explants necessary for 3 dishes).
a. Prepare 1 plastic Petri dish containing 1x PBS to collect the embryos, and keep it on ice.
b. Euthanize the pregnant female in accordance with the approved protocol validated by the Ethics Committee. In our case, pregnant female Swiss mice were euthanized by cervical elongation at embryonic day 13.5 (E13.5).
c. Extract the uterine horns containing the embryos from the pregnant mouse ( Figure 1A), and collect them in the PBS-containing plastic Petri dish.   ii. Invert the head to see the under side ( Figure 2C). At the E13.5 stage, the palate is not completely closed, and this orientation allows direct observation of the septum separating the right and left nasal cavities.
iii. Cut the lateral sides of the developing palate in the horizontal plane ( Figure 2C). Once this is done, under the microscope it is possible to see the olfactory mucosa covering this cavity and attached to the septal, dorsal and lateral cartilages.
iv. Make a coronal section of the tissue through the septum and the turbinate bones ( Figure 2C).
v. Detach the caudal part of OP1 from the olfactory bulb and cut beside the turbinates ( Figure 2C, line). ii. Use forceps to maintain them immobile, and cut the described below pieces (hereafter referred to as OP2s) using the microscalpel ( Figure 2D). x. Incubate culture medium (Solution B) at 37 °C in a water bath.
xi. Transfer some OP1 into a glass Petri dish, gently isolate the OE tissue from the lamina propria and cut the OE sheets using the micro-scalpel into small pieces of about 50-150 μm diameter; these are the final explants ( Figure 2F).
xii. Store explants in a clean well containing L-15; 5% FBS on ice.
xiii. Once laminin coating is finished (after minimum 3 h at 37 °C, 5% CO2), remove the dishes from the incubator. xiv. Remove the laminin (using a pump aspirator or a pipette) and replace it directly by the appropriate volume of culture medium (6 ml in a 50 mm diameter dish). 10. Arrangement of explants in the dish a. Collect 5 to 6 explants using a 10 µl Pipette + tip and transfer them one by one directly to the appropriate position.
b. Place the tip as close as possible to the glass surface without touching it (contact would damage the coating). Explants should slowly descend by gravity.
c. Make sure there is at least 1 mm between 2 explants (see Figure 2F for scale). We usually put 5 to 6 explants per dish, forming a cross in the center. The bead density can vary from one batch to another. Our batch had a concentration of 6.826 x 10 7 beads/ml. Using a 1/5,000 dilution of these beads, we had a final concentration of 1.36 x 10 4 beads/ml in each dish, which was to a satisfactory density. a. First, prepare the Strepta-Beads solution: Note: The 1/5,000 dilution of the beads is performed in 2 steps.
i. Vortex the beads.

Quality control of the axon biotinylation:
In order to check that axons are properly biotinylated, and able to be bound by beads, a quality control can be performed. This quality control consists of detecting biotin using streptavidin, and to verify that beads can bind to axons.  The principle, tools and devices used in our experiments are described in details and illustrated by remarkable figures in Gourier et al., 2008. Therefore, in the protocol below, we will not describe the aspects that are already detailed in that technical publication. We will, however present, the detailed protocol and parameters used for tension measurement of olfactory axons.

Micropipette preparation
Micropipettes are prepared as described in Gourier et al. (2008), as follows: a. Elongate a borosilicate glass capillary using the micropipette puller.
b. Using the microforge, as described in Gourier et al. (2008), adjust the micropipette diameter to give an aperture (inner diameter) of about 2 µm, to optimize the holding of the red blood cell by the pipette. h. Check that the manipulated bead remains attached to both the axon and RBC, by slowly moving the pipette backwards and forwards. If the bead detaches from either the axon or the RBC, repeat the procedure with another bead attached to an axon.
i. In the favorable cases where the bead remains attached to both the axon and the RBC, proceed with the acquisition of data for tension measurement as follows: i. The whole manipulation should be continuously recorded using the CCD camera.
ii. Pull the micropipette a short distance, induce elongation of the RBC; this often results in lateral deflection of the axon. An equilibrium is rapidly reached between the force induced by the probe and the transverse projection of the reaction force of the axon axial tension.
iii. Pull the micropipette again, a bit further, to induce an additional deformation of both the RBC and axon (a second equilibrium is reached).  (2017a and 2017b).

Data analysis
The video data obtained using this protocol allow the extraction of two key parameters necessary to calculate axon tension: 1) the deformation of the RBC (used here as a force transducer), allowing the calculation of the force (F) with which the bead (and hence the axon) is manipulated; and 2) the deflection of the axon, allowing the calculation of axon tension as a function of the applied force F. The procedures for assessing these two key parameters, as well as to calculate axon tension, are detailed in one software technical paper (Šmít et al., 2017b), and in a research paper (Šmít et al., 2017a), both in open access. Therefore, the data analyses will not be described further here. Because Poly-D-Lysine (PDL) is thought to be less toxic for the cultures, we performed experiments using PDL 10 µg/ml, and observed that it is suitable for explant adhesion to the coverslip.
2. PBS 1x 0.06% Glucose can be used for embryo dissection to reduce costs but L-15 medium preserves viability and must be used at least for explant storage.
3. The microscalpel allows a nice cutting edge, preserves the integrity of the explant and reducing cell death. If this tool is not available, replace it with a 26 G needle at the tip of a 1 ml syringe. 4. The EZ-Link Sulfo-NHS-SS-Biotin is also available by weight (100 mg). We find that using the No-Weigh Format enhances reproducibility.