GeneMedi's protocol / procedure for the diagnostics application-FACS
1 Flow cytometry / Fluorescent activated cell sorting
Each cell expresses hundreds of thousands of cell surface antigens that specify their cell type, biological function, development stage, and much more. Cells residing in different organs have characteristic cell surface antigens, and the determination of these cells using the specific fluorochrome-conjugated antibodies can be analyzed by flow cytometry. Titration for flow cytometry is used to determine the antibody amount and concentration resulting in the highest signal of the positive population along with the lowest signal of the negative population. It is also the best way to eliminate nonspecific binding of an antibody.
Flow cytometry is a powerful tool because it allows simultaneous multiparametric analysis of the physical and chemical characteristics of up to thousands of particles per second. This makes it a rapid and quantitative method for analysis and purification of cells in suspension. Using flow, we can determine the phenotype and function and even sort live cells.
FACS is an abbreviation for fluorescence-activated cell sorting, which is a flow cytometry technique that further adds a degree of functionality. By utilizing highly specific antibodies labeled with fluorescent conjugates, FACS analysis allows us to simultaneously collect data on, and sort a biological sample by a nearly limitless number of different parameters. Just like in conventional flow cytometry, forward-scatter, side-scatter, and fluorescent signal data are collected. The user defines the parameters on how cells should be sorted and then the machine imposes an electrical charge on each cell so that cells will be sorted by charge (using electromagnets) into separate vessels upon exiting the flow chamber. The technology to physically sort a heterogeneous mixture of cells into different populations is useful for a wide range of scientific fields from research to clinical. Nowadays the terms "flow cytometry" and "FACS" are often used interchangeably to describe this laser-based biophysical technique.
1.1 Antibody titration protocol using 96 well plates
(1) Prepare cells at a final concentration of 2.5×104 per µl in blocking buffer
(2) Aliquot 40 µl of cells per sample
- If using a viability dye, make sure to include a single stained control for viability (for compensation)
(3) Prepare the dilution series
- Prepare the concentration so that 10 µl can be added to each sample
(4) Add the antibody to the cells and gently mix.
(5) Place on ice, in the dark, for 20 minutes
(6) Add ~300 µl staining buffer to each well
(7) Centrifuge the plate at 900xg
(8) Aspirate (or Flick) the solution out of the wells
(9) Repeat steps 6-8 two more times
(10) Resuspend the sample in ~200 µl final volume
- If using viability dye, add to the sample
- If sample has to be fixed, resuspend in~200 µl of fixation buffer
- If using fixation buffer, cell impermeant viability dyes (PI, 7AAD, DAPI, etc) cannot be used
- Collect sufficient events for analysis (10,000 positive events)
(11) Analyze the cells on the flow cytometer
(12) Export the data and analyze in software of choice
(13) Calculate the Staining Index (SI) and plot versus concentration of antibody3
- Staining Index (SI) = ((median pos-median neg) / ((84%neg-median neg)*0.995)
1.2 Protocol for cell sorting
(1) Remove spleens, LN, etc. into media on ice. Disrupt into single cell suspension using your favorite technique and pass through 70uM filter.
(2) Centrifuge at 1500 RPM for 10 minutes at 8˚C.
(3) Perform red blood cell lysis. Re-suspend in FACS staining buffer. (Use this buffer also for all washes until directed to use Sorting Buffer.) Adjust cells to 20-50 * 106/ml for typical staining reactions.
(4) Add the appropriate number of cells to be stained into a FACS tube or 15mL conical tubes.
(5) Block with Fc Block (2.4G2). Incubate on ice for 15 min. Centrifuge at 1500 RPM for 10 minutes at 8˚C and pellet.
(6) Remember to save some cells to stain single color controls for the set-up of the machine. You must have enough unstained cells and cells stained with each color used in order to set up the machine. Preferably use brightly staining reagents that stain a high percentage of cells, such as B220 or Thy1 for mice.
(7) Add antibodies to stain the cells that will be sorted; use at 1x or 0.5x of the typical concentration used to stain cells for analysis. Incubate for 20-30 min on ice. Fill tube with media up to top. Spin 10 min. @ 1500 RPM, 8˚C, remove supernatant and re-suspend pellet.
(8) Stain with secondary reagent, if needed, for 20 min. on ice. Wash as before.
(9) Wash once more with Sorting Buffer. Cells must be in low protein buffer (low FCS or BSA) to prevent the sorters from clogging.
(10) Re-suspend cells at a concentration of 20-50×10^6/ml. This will ensure a sorting speed of approximately 18,000-20,000 events per second at the optimal pressure. Filter again through 70uM filter, again to prevent clogging.
(11) Collection tubes can be blocked with FCS prior to adding collection medium. Collection medium can be supplemented with additional serum to offset the sheath buffer that will dilute the collection medium as cells are collected. Optimal conditions need to be worked out individually depending on the cells sorted, etc. Cells can be collected in either FACS tubes or 15 mL conical tubes.
picture1
Principle of fluorescence-activated cell sorting (FACS) (Adopted from: Mehanna, Radwa. (2017). Physical versus Immunological Purification of Mesenchymal Stem Cells. 10.5772/intechopen.69295.)
1.3 Advantages of flow cytometry
(1) High throughput analysis of large cell populations
(2) Multiparameter analysis allowing for measurement of multiple parameters simultaneously, including cell surface markers and intracellular signaling molecules
(3) Rare cell detection allowing for the identification of rare cell populations
(4) Cell sorting enabling the isolation of specific cell populations for downstream analysis
High sensitivity and specificity in detecting rare cell populations or molecules
(5) Real-time analysis with immediate results
1.4 Disadvantages of flow cytometry
(1) Expensive equipment and reagents can be a barrier to use
(2) Requires specialized training and expertise
(3) Limited by the need for single cell suspensions, which can be challenging for certain cell types or tissues
(4) Sensitivity can be affected by background noise or autofluorescence
(5) Limited capacity for high resolution imaging
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