Since this method analyzes cells from solution, it can be used to study cells in a near native environment. This includes analysis of patient derived cell samples. This technique requires little to no sample preparation of cells.
Therefore, we can analyze individual cells online under ambient conditions, which allows us to explore cellular heterogeneity. This technique can be applied to study liquid biopsies from a variety of different disease states, since it can analyze different types of cells directly isolated from patient samples. To convert single bore glass tubing into a tapered probe with a sharp tip, first place a single bore glass tube into the clamps of a vertical pipette holder, centering the glass with respect to the heating coil and tighten to secure the tube in place.
The heating coil is comprised of an 18 gauge nickel chromium resistance wire coiled around a metal rod 2.5 times. Set the glass tubing with temperature program 19.5. Set the solenoid plunger at four.
Trigger the solenoid to pull the glass tubing. This step creates two probes fused at the tip. Use sweaters to cut approximately one millimeter away from the tip of each probe, creating an orifice of approximately 10 microns in diameter at the probe tip.
To bend the glass probe for easy coupling to the ICMP, single probe SCMS set up. First set a pulled glass probe into the microforge. Position the top approximately three millimeters above the platinum heating wire.
Then, turn the heat on the platinum wire to 30%of the maximum temperature. Bend the probe approximately 45 degrees from the original position. Place the inverted microscope microinjector in two cell manipulation systems on a motorized table for easy coupling with the mass spectrometer.
To set up the glass cell selection device, insert the glass cell selection probe inside the metal holder of the microinjector by placing the long slide into the capillary holder and tightening the screw to secure the probe in place. Position the probe tips angled parallel to the heated plate. Secure the metal holder of the micro injector into the cell manipulation system.
Then position the probe tip near the middle of the inverted microscope light. Secure the glass slide containing the single probe into the arm clamp of the cell manipulation system. Connect the solvent providing capillary to a conductive union by placing the capillary into the sleeve of the plastic ferrule and finger tightening the fitting.
Connect the other side of the conductive union to a capillary which is connected to a syringe containing the sampling solvent by placing the capillary into the sleeve and tightening the fitting. Use acetonitrile with 0.1%formic acid as the sampling solvent in these experiments. Secure the syringe into the syringe pump on the mass spectrometer.
Position the nanoelectrospray ionization or nano ESI emitter approximately one millimeter to the orifice of the extended ion transfer tube. Use the cell manipulation system to control the spacial movements of the single probe and position the nano ESI emitter centrally in front of the extended ion transfer tubing. Pipette the cells from the cell culture flask into a 15 milliliter centrifuge tube.
Spin the cells at 400 times G in 37 degrees Celsius for five minutes and discard the supernatant. Resuspend the cells in four milliliters of RPMI medium containing the drug compound at the desired treatment concentration. Customize the experimental parameters for the mass spectrometer.
Under the scan mode heading of the instrument software, select define scan. Use a resolution of 60, 000 at M over Z 400, one microscan 100 millisecond maximum injection time and automatic gain control on. Under syringe pump, select the flow rate of 150 nanoliters per minute.
Select NSI source and apply a voltage of approximately 4.5 kilovolts. Turn on the inverted microscope at 40 times magnification selected for both the top plate and bottom lens. Connect it to the USB port of a laptop to capture live video feeds.
Turn on the heated plate and set it to 37 degrees Celsius. On the computer, go to the acquire data tab and select continuously under acquired time. To prepare the sample for analysis, pipette two to three of the sample into the lid of a small petri dish.
Position the sample in the center of the light from the inverted microscope on top of the heated plate. Prepare the glass cell selection probe for analysis. Use the cell manipulation system to move the probe so its top is focused under the inverted microcsope in the same plane as the cells.
Use the cell manipulation system to move the cell selection probe tip to a targeted cell for analysis. This process is monitored using the inverted microscope. Gently turn the handle of the microinjector to adjust the position of the mineral oil inside the tubing.
A gentle suction is provided by the microinjector to secure the targeted cell to the cell selection probe tip. Use the cell manipulation system to move the cell at the cell selection probe tip to the single probe tip using a digital microscope focused on the single probe tip to monitor this process. Untreated K-562 cells are used to establish the experimental method.
In a typical SCMS experiment, obvious changes of mass spectra can be observed from transferring a cell during the detection of cellular contents and after finishing the measurement. Three common cellular lipid peaks including PC 34:4, PC 36:4, and PC 38:5 are monitored to ensure the cell is successfully transferred and cellular contents are detected. The identity of many PCs in the mass range of 750 to 850 are confirmed using MSMS on untreated cell lysate samples.
Using the ICMP single probe MS set up, Gemcitabine, Taxol and OSW1 were detected following incubation with K-562 cells. These results suggest this method can be used to study intracellular lipids, drugs, and metabolites on the single cell level from cells in solution in a near native environment. Remember to securely place the glass cell selection probe into the universal pipette holder so that suction can be adequately applied.
This method can also be applied to patient derived samples to distinguish differences among cellular metabolites from a variety of cell types, which allows us to gain a better understanding of these disease states. After developing this method, we could potentially implement the quantification of drug compounds at the single cell level. When working with cells, it is important to have appropriate personal protective equipment including lab coat and gloves.
Additional eye wear may be used when working with glass.
