Single Cell Volume Measurement Utilizing the Fluorescence Exclusion Method (FXm)

Materials and Reagents

A.  Microfluidic Supplies

A thin microfluidic channel is required. This channel is usually constructed out of a sandwich between a polydimethylsiloxane (PDMS) top and a glass coverslip bottom; however, two glass coverslips separated by adhesive tape have also been used (Bottier, 2011). We will cover the use of PDMS in this protocol. The stiffness of the bottom may also be modified by coating with a thin layer of PDMS. Following this protocol, to include the manufacture of custom PDMS chips (which may alternatively be purchased (Bottier, 2011), the following materials will be required:

1. Printed mask, (2D) negative of the mold (FineLineImaging, self-designed, may be designed with AutoCAD)
2.  PDMS for the chip (Sylgard 184)
3. 12.6 kPa PDMS for coating (Dow Corning Toray, catalog numbers: CY52-276A and CY52-276B for 12.6 kPa) (Bergert et al., 2016)
4. 3 kPa PDMS for coating (Dow Corning Toray, catalog numbers: CY52-276A and CY52-276B) (Style et al., 2014)
5. 0.4kPa PDMS for coating (Quantum Silicones, catalog numbers: QGel 920A and QGel 920B) (Gutierrez et al., 2011)
6. Glass substrate (50-mm glass-bottom Petri dishes, FlouroDish Cell Culture Dish; World Precision Instruments)
7. Blunt-tipped 21-gauge needle and syringe (McMaster Carr, catalog number: 75165A681)
8. Isopropyl alcohol (IPA)

B.  Cell Culture Supplies

Culture media and supplies are largely standard with the major exception being the fluorescent dye. The dye must be conjugated to something which is sufficiently membrane-impermeable and water soluble so to be uniformly distributed throughout the culture media and excluded from the cell. We tried a variety of conjugates and settled on a large dextran group.

1. DMEM (Corning)
2. PBS (phosphate-buffered saline solution)
3. Penicillin/streptomycin (Gibco)
4. FBS (Sigma-Aldrich)
5. Matrix Protein (50 μg/ml of type I rat-tail collagen) (Corning, catalog number: 354236)
6.  Alexa Fluor 488 Dextran molecular weight 2,000 kD (Sigma-Aldrich, catalog number: FD2000S-1G)

Equipment

1. Silicon wafers (Wafer World, catalog number: 2886)
2. Photoresist (SU8-3000 and developer, Kayaku Advanced Materials)
3. Oven (VWR, Gr Con 2.3CF)
4. Plasma cleaner (Harrick Plasma, pdc-32g)
5. Microscopy/Cell Incubation
   The method below utilizes three imaging modalities: confocal, epifluorescence, and differential interference contrast (DIC) microscopy. Only an epifluorescence microscope is essential for this technique; however, we will emphasize the value of a confocal scope for the measurement of microfluidic channel height during device calibration. Other transmitted light images (e.g., phase contrast) may be substituted for the DIC images in this protocol, though we expect some steps to be easier to implement with DIC. Standard cell incubation equipment is required, dependent on the cell type.
6.  Microfabrication
   We manufactured our PDMS chips using standard photolithographic equipment; however, due to the relatively large feature size, devices may be assembled to a satisfactory quality outside of a clean room (e.g., a biosafety level 1 space is appropriate). Additionally, this device is a single layer and thus its construction does not require a mask aligner. A simple UV lightsource, spin-coater, and hot plate should be sufficient for mold construction. Bonding of the substrate and PDMS chip is achieved by exposing one or both components to oxygen plasma. A variety of plasma sources may be used. An oven is useful for quickly curing PDMS and ensuring bonding of the PDMS chip to the substrate; however PDMS will eventually cure/bond at room temperature. A sonicator is useful for cleaning the PDMS chips before they are bonded to the substrate but this step may be skipped if necessary.
   

Software

1. MATLAB (MathWorks)
2. ZEN 3.0 (Zeiss)
   

Procedure