Fluorescence Activated Cell Sorting (FACS)

The successful multi-channel FACS relies on the clean separation of the spectrum of fluorescent reporters that are co-expressed in the same cell population. Thus, this requires careful selection of reporters and multiple single or dual color control samples to calibrate the gate and compensation conditions. The parameters given below were determined for a BD FACSAria^™ flow cytometer equipped with a 488-nm blue laser, a 561-nm yellow/green laser, a 633-nm far red laser and a 407-nm ultra violet laser.

Four single color samples are prepared for calibration of gating and compensation on flow cytometry sorter

None-electroporated sample (no fluorescent reporter)

Single color sample 1- electroporated with MyoD905>GFP

Single color sample 2- electroporated with Mesp>tagRFP

Single color sample 3- electroporated with Hand-r>tagBFP

Three dual color samples to test the separation of channels as pair

Dual color sample 1- electroporated with MyoD905>GFP and Mesp>tagRFP

Dual color sample 2- electroporated with MyoD905>GFP and Hand-r>tagBFP

Dual color sample 3- electroporated with Mesp>tagRFP and Hand-r>tagBFP

One triple color sample electroporated with MyoD905>GFP, Mesp>tagRFP and Hand-r>tagBFP to test the separation of all the three channels

Before loading the samples into the sorter, filter the cell suspension again on the cell-strainer cap of a 5 mL round-bottom tube and gently pipette the cell suspension 3–5 times.

Load the non-electroporated sample into the sorter and count ~10,000 cells. Distinguish the well dissociated single cells from undissociated tissues and debris based on size and internal complexity which can be defined by forward scatter (FS) and side scatter (SS). Gate the single cell population as P1 (Fig. 2a).

Samples electroporated with blank buffer, Mesp>tagRFP, MyoD905>GFP and Hand-r>tagBFP fluorescent reporters are used to gate the TVC progeny populations. a. Gate P1 for single cell population based off the FS and SS from the blank sample. b. Gate the tagRFP⁺ cell population using the sample electroporated with Mesp>tagRFP. c. Gate the GFP⁺ cell population using the sample electroporated with MyoD905>GFP. d. Gate the tagBFP⁺ cell population using the sample electroporated with Hand-r>tagBFP. e. Define the cells only have tagRFP as P2 in the sample co-electroporated with Mesp>tagRFP, MyoD905>GFP and Hand-r>tagBFP. f. Analyze P2 with gates set for tagRFP and tagBFP, define the tagRFP-tagBFP⁺ cell population as P3. P3 is TVC progeny that we sort for downstream genome-wide analysis.

Load the Single color sample 1 into the sorter and count ~10,000 cells. Gate the GFP+ population using 488-nm blue laser, PMT (Photomultiplier Tubes) Detector E, LP (Long Pass) Filter 505 and BP (Band Pass) Filter 530/30 for FITC (Fig. 2b).

Load the Single color sample 2 into the sorter and count ~10,000 cells. Gate the tagRFP+ population using 561-nm yellow/green laser, PMT Detector B and BP Filter 586/15 for DsRed (Fig. 2c).

Load the Single color sample 3 into the sorter and count ~10,000 cells. Gate the tagBFP+ population using 407-nm (violet) laser, PMT Detector B and BP Filter 450/50 for Pacific Blue (Fig. 2d).

Mix the Single color sample 1 and 2 by 1:1 and load the sample mixture into the sorter, and count ~10,000 cells.

Adjust the compensation, ensure clean separation of the GFP+ and tagRFP⁺ populations.

Repeat the same steps using mixtures of single color samples 1 and 3, and 2 and 3, adjust the compensation to ensure the clean separation of GFP⁺ vs. tagBFP⁺ populations and tagRFP⁺ vs tagBFP⁺ populations.

Mix the Single color samples 1, 2 and 3 in 1:1:1 proportions. Load the sample mixture into the sorter and count ~10,000 cells, making sure the GFP⁺, tagRFP⁺ and tagBFP⁺ cell populations are still well separated from each other.

Load the Dual color sample 1 and count ~10,000 cells. Use the GFP⁺ and tagRFP⁺ gate, which was defined by single color sample to identify the cell population with only GFP expression, only tagRFP expression and with both GFP and tagRFP expression. Gate the GFP-tagRFP⁺ population.

Repeat the same steps for 2-color samples 2 and 3, define the gates for GFP-tagBFP⁺ and tagRFP-tagBFP⁺ populations.

Note: The calibration of gating (step1-step10) is recommended every time before sorting. However, the BD FACSAria^™ flow cytometer has been proved to be consistent and repeatable across daily operations. Therefore, the calibration of gating might be skipped when sorting the same type of samples frequently in a short period, such as ONE month.

Load the triple color sample and count ~10,000 cells.

Analyze the sample with the lasers and filters for FITC and DsRed, select the population with only tagRFP signal, mark it as P2 (Fig. 2e).

Analyze the P2 population with the lasers and filters for DsRed and Pacific Blue. Select the cell population that falls into the tagRFP-tagBFP⁺ gate and give it the P3 identification (Fig. 2f).

In our experiments, this P3 population consists of TVC progeny which co-express tagRFP and tagBFP (co-selection) but without the expression of GFP (counter selection of mesenchyme cells with leaky Mesp>tagRFP and Hand-r>tagBFP expression). These cells only represent 0.1%–0.2% of the P1 population from the whole embryo.