20211101 Restart

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Restart of project

New microscope glass, microfluidic preparation

On Oct 28 2021 Anniken, Adam, Endre and Thomas went to Lab 128 with the intention of resuming the microfluidic incubator project. PDMS had been prepared in advance. The purpose of meeting was to test adhesive glass slides in the hopes that they would, electrostatically and/or chemically, attract our cells and anchor them to the slides. Test 1 consisted of plasma treating the PDMS but not the adhesive slide. Test 2 consisted of plasma treating both the PDMS and the adhesive slide. Initially, both tests seemed to generate microfluidic chips where the PDMS was properly attached to the slides.

New microscope glass, cells for test

After having assembled the microfluidic chips, we prepared our cells in order to inject them into the chips.

The chip from test 1 did not withstand the injection of cells. The PDMS detached from the adhesive slide more or less immediately after being handled.

The chip from test 2 did withstand the injection of cells and the PDMS did not detach from the adhesive slide. We started a timelapse to study the cell adhesion on this slide.

On Oct 29 2021 Anniken's update on the timelapse. The cells did not adhere, but the PDMS was still properly attached to the glass slide. Plasma cleaning the adhesive slide might have disturbed the cell-adhesive properties. Since we don't know how or with what the adhesive slides are treated, we will move on to trying fibronectin as a coating agent instead.

Suggestions for continuation using bovine fibronectin

Anniken has found a protocol for coating with fibronectin and an alternative protocol.

The first recommends 2-10 mg/cm^2, the alternative protocol recommends 1-5 ug/cm^2. We need Tris buffer but decided to try PBS instead. Balance and weighing equipment is found in lab 420.

We have decided to aliquot the fibronectin into smaller vials in order to freeze them and make the fibronectin last. The 1 mg of fibronectin will have to be reconstituted and diluted with buffer before being divided into vials so that the volumes can be handled precisely. After reconstituting and diluting the fibronectin, we apply a thin coating to the microfluidic chips. The fibronectin needs time to incubate at room temperature in order for the proteins to solidify and for the functional domains to interact properly with each other and the glass slide.

Fibronectin coating protocol

Reconstitute lyophilized fibronectin in suitable solvent at 1 mg/mL:

Amount of solvent needed to reconstitute 1 mg of fibronectin at 1 mg/mL:

1 mg/mL = 1 mg fibronectin/1mL destiller water

Dilute to desired concentration of 250 ug/mL with physiological buffer:

c1V1 = c2V2

V2 = c1V1/c2

V2 = (1 mg/mL * 1 mL)/(250 ug/mL)

V2 = 4 mL

The final volum (4 mL) consists of 1 mL reconstituted fibronectin and 3 mL physiological buffer.

Determine amount of fibronectin needed for culture vessel with a total surface area of 5 cm^2:

Recommended amount of coating: 5 ug/cm^2

Mass of fibronectin: 5 ug/cm^2 * 5 cm^2 = 25 ug

Volume of diluted fibronectin:

V = m/c = 25 ug/250 ug/mL = 0.1 mL

The volume of diluted fibronectin needed for each culture vessel is 0.1 mL.