I am new to FACS field. I am planning to have two surface markers targeted to sort one kind of cell via FACS. Is it possible? Can I have some protocol/links from you guys with experience :) Thank you very much!
Dear Aparamita!
It's possible .
Please You read the article:
Article Flow Cytometry Protocols for Surface and Intracellular Antig...
For example, anather protocol:
Assessment of cell viability using DNA-binding dyes
YO-PRO-1 and PI by flow cytometry
Cell staining with fluorescent DNA-binding dyes YO-PRO-1 and propidium iodide (PI) was used to determine apoptotic or necrotic types of tumor cell death upon exposure to NGF, PG-1 LL-37, and their combinations with chemotherapy drugs. Propidium iodide binds to DNA due to intercalation between guanine-cytosine nucleotides, after which its fluorescence increases 20–30 times with an excitation maximum at 535 nm and emission maximum in the red region (617 nm). Propidium iodide is unable to penetrate through intact membranes of living cells; therefore, it can accumulate in cells either at the late stages of apoptosis, when secondary necrosis begins, or in the case of an initially necrotic variant of their death.
To assess the early death of cells with a damaged membrane, we used the green fluorescent DNA marker YO-PRO-1 (YP1). YO-PRO-1 has a large size (630 Da), which prevents its penetration through the membrane of living cells. However, during apoptosis, when the integrity of the membranes is disturbed, YO-PRO-1 penetrates into cells and binds to DNA, after which the fluorescence of the dye increases with a maximum of excitation and emission at wavelengths of 491⁄509 nm . Thus, the combined use of propidium iodide and YO-PRO-1 makes it possible to differentiate cells that entered early apoptosis (green fluorescence of YO-PRO-1) from cells in the late stages of apoptosis or necrosis (red fluorescence of propidium iodide).
A suspension of C6 and U251 gliomas cells at a concentration of 1 × 106 cells / ml was used in the experiments. The sample volume for flow cytometry was 500 μL. A control of intact cells was also prepared, into which the corresponding volume of PBS was added instead of the tested preparations. Experimental samples and control were incubated for 1 day in a CO2 incubator at 37 ° C. After incubation, the cells were pelleted by centrifugation at 1000 g for 10 minutes and resuspended in 500 μl of PBS. Staining of YO-PRO-1 and PI cells was performed in tubes for cytometric counting 12x75 mm (Beckman Coulter, USA). For this, 5 μl of 20-fold YO-PRO-1 working solution (Invitrogen, USA, final concentration 250 nM) was added to 100 μl of cell suspension (1 × 106 cells / ml). The working solution was prepared ex tempete by adding 190 μL of PBS to 10 μL of a stock solution (100 μM DMSO). Then, 10 μl of PI solution (Sigma-Aldrich, USA, final concentration of PI 1 μg / ml) was added to the samples. Coloring was carried out at room temperature for 15 minutes in a dark place. Upon completion of incubation, 200 μl of PBS was added to the samples and analyzed on a Navios ™ flow cytometer (Beckman Coulter, USA). For each of the samples, at least 50,000 single cells were analyzed. To distinguish single cells from aggregates and subsequently to discriminate aggregates from the analysis, we used the following signal combinations for direct (a value proportional to cell size) and lateral (a value characterizing cell structure) light scattering - the intensity of the peak versus the intensity of the integral signals by FS or SS, and also flight time against the intensity of the FS or SS integral signals. The analysis of the obtained results was carried out using the Kaluza ™ software (Beckman Coulter, USA),
Definitely you can do. We are doing more than 10 markers on a cells.
You can use multiple markers to identify your population, use gating strategy to sort.
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