Jan 21, 2022

Public workspacemPSM protocol V.2

This protocol is a draft, published without a DOI.
  • 1EMBL
Takehito Tomita
Icon indicating open access to content
QR code linking to this content
Protocol Citation: Takehito Tomita 2022. mPSM protocol. protocols.io https://protocols.io/view/mpsm-protocol-b35dqq26Version created by Takehito Tomita
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: January 21, 2022
Last Modified: January 21, 2022
Protocol Integer ID: 57221
Abstract
Protocol to perform 2D ex vivo segmentation assay (Lauschke et al. 2013).
Coat dishes/plates with fibronectin.
1h
Dilute fibronectin (Sigma, Cat.No: F1141) with PBS to 50ug/mL (20 fold dilution).
Fill each well of 8-well plate (NuncTMLab-TekTMII Chambered Coverglass, Cat.No: 155409PK) with ~160μl of fibronectin solution and incubate for 1 hour in 37°C.
Remove all fibronectin solution from the well(s) and dry for 1 hour at room temperature (~25°C).
Prepare dissection medium and culture medium.
10m
Mix the following ingredients in a 50 mL falcon tube:

Amount0.4 g BSA
Amount400 µL glutamine
Amount35.52 µL 45% glucose
Amount40 mL DMEM-F12
Vortex well.
Separate Amount10 mL from the mix in step 2.1 and filter through a 0.22μm filter (Millex-GV, Cat.No: SLGV033RS) into a fresh 15 mL falcon tube. Add Amount100 µL Penicillin/Streptomycin (Gibco, Cat.No: 15140-122) to achieve
100U/mL. This mix is used as culture medium. Equilibrate in 37°C 5% CO2 until use.
Add Amount510 µL HEPES to the remaining Amount30 mL mix in step 2.1. This mix is used as dissection medium. It can be kept at the bench until use.

Wash the dried wells with ~200μL culture medium per well. Apply fresh culture medium (160uL ~ 300μL per well) and equilibrate in 37°C 5% CO2 until use.

Extract mouse embryos and collect tails in dissection medium.


20m
Cut the tail bud using a scalpel in dissection medium. Be careful not to flick the tail buds as this damages the tissue.
5m
Wash the tail buds once in a dish containing culture medium. Quickly move the tail buds to the fibronectin coated wells. Align the tissue orientation using forceps to place the cut side downward.
1m
Incubate in 37°C 5% CO2 for 1hr to let the tissue attach.


1h
Place the well into the metal box to fit into the microscope. Add a strip of wet paper towel inside the metal box to prevent drying. Carefully move the box into the microscope, as tissue can detach quite easily.
Set up imaging.