How does it work?
Cell sorting is a technology to isolate and purify one or more cell population(s) from a heterogeneous single cell suspension. The sorting process is based on predefined criteria like cell size, shape (morphology), and surface and/or intracellular protein expression. Flow cytometric cell sorting is used in a wide field of applications like physiological research, protein engineering, cell engineering, and for medical purposes like cell therapy (autologous adoptive cell transfer "ACT") and transplantation.
The most widely used type of sorting is the electrostatic cell sorting. The fundamental process of this technique is the formation and charging of droplets. The majority of cell sorters use a piezoelectric device to form droplets by the application of an acoustic wave in the flow cell of the instrument. By that means droplet formation is extremely fast and stable with a predictable rate.
For most electrostatic cell sorters, cells are transported through the system in a stream of sheath fluid. Based on the principles of hydrodynamic focusing, cells are lined up like beads on a string in direction of the flow. At the interogation point cells are hit by laser light and the resulting optical signal is collected and processed by optical and signal processing systems. When a target cell is detected, a charge is applied to the droplet that contains the cell of interest. All droplets, charged or uncharged, then pass through an electrical field that is generated by the deflection plates. Charged droplets are pulled towards the plates, away from the uncharged droplets, and fall into collection tubes, forming side streams, while uncharged droplets pass into the waste.
Major difference between AriaIII and MoFlo Legacy
The Core Facility works with a BD FACSAriaIII and a Beckman Coulter MoFlo Legacy which are both charged droplet sorters. The fundamental difference between the two systems lies in the location of the interogation point.
For the FACSAriaIII cells are forced through an optically gel-coupled cuvette flow cell to interogate with the laser(s). This cuvette based interrogation gives a better optical resolution to distinguish dimly positive populations from background fluorescence. In addition scatter performance is optimized for resolving lymphocytes, monocytes, and granulocytes.
MoFlo Legacy is a Jet-in-Air system meaning that the interogation of the cells with the laser(s) happens "in the air". Scatter and fluorescence signals are collected by a microscope objective that is focused on the interogation point. By using a microscope objective less light is collected and therefore sensitivity and resolution will always be suboptimal in comparison to a cuvette based system.
Which sorter is best for my cells?
The two types of technologies cuvette based and jet-in-air have advantages and disadvantages. No cell sorter design can fit all needs, especially when the cell type to be sorted is sensitive to manipulations. For some more sensitive cell types like DCs an increased cell death and a loss of function can be observed after the sorting process. This effect might be different for the two cell sorters operated by the Core Facility. Most probably responsible for this are differences in fluid dynamics through the sample path that may damage the cells and decrease their ability to function. Since the two different technologies are available in the Core Facility it would be optimal to perform a test sort on both systems in order to find out which of the two is best for the cells of interest.
Choosing the right nozzle!
The cell sorters can be operated with different nozzle sizes. The size of the nozzle depends on the dimensions of the cells to be sorted. As a general rule of thumb: The cell size should not exceed one-fifth of the nozzle diameter. The table below illustrates the sorting conditions related to the different nozzle sizes.
|Nozzle size (um)||70||85|
|Pressure (Psi)||60 - 70||45||20||12|
|Max. cell size (um)||14||17||20||30|
|Max. events/sec||22.000 - 30.000||11.000||10.000||5000|
|Cells/ml of sample||3 - 5 x 107/mL||1 - 2 x 107/mL||5 - 7 x 106/mL||1 - 2 x 106/ml|
|~ Drop Size (nl)||1||2||4||12|
|Cell/ml Post-Sort||1 x 106/mL||0.5 x 106/mL||2.5 x 105/mL||1.2 x 105/ml|
Using the wrong nozzle size causes fanning of the side streams, poor sort purity and nozzle clogging.
Cell sorting takes time. The time that is needed to sort the desired number of your target cells is dependent among others on the nozzle size, the sort mode (enrich versus purity), percentage of target cells in the sample, and the sample itself. The following table should help to estimate the sort time.
|Time needed to sort required number of target cells at 10.000 cells/second|
|number of cells wanted||desired number of cells as % of total cells|
|103||2 min||10 s||2 s||1 s||0.2 s|
|104||17 min||2 min||20 s||10 s||2 s|
|105||47 min||17 min||3 min||2 min||20 s|
|106||27 h||47 min||33 min||17 min||3 min|
|107||11 d||27 h||5 h||47 min||33 min|
|108||115 d||11 d||2 d||1 d||5 h|