The overall goal of this spectral cytometry technique is to distinguish different fluorochromes using their entire emissions spectra. More over this protocol enables implementation of autofluorescence as an independent parameter, allowing for a proper analysis of cells isolated from solid organs. This method can help answer key questions in the fields immunology and development of stem cell biology such as how to identify rare cell populations.
The main advantage of this technique is it's unique capacity to simultaneously analyze a large number of parameters, and to manage the autofluorescence of solid tissues. All though this method is used for preparation of a single cell suspension derived from intestine and heart, it can also be applied to other organs, such as lung, skeletal, muscle, liver, fat, and kidney. To begin, isolate and wash the small intestine from an adult mouse.
Then, place the tissue on a paper towel, and using sharp scissors, carefully remove the Peyer's patches. Open the intestine by cutting it longitudinally and divide it into one centimeter pieces. Then, transfer the tissue to a beaker filled with 30 milliliters of HBSS supplemented with ten percent FCS, and incubate it at 37 degrees Celsius with constant stirring for 30 minutes.
Once the incubation is completed transfer the cell suspension to a 15 milliliter plastic tube and vigorously vortex it for five minutes. Then, incubate the suspension on ice for ten minutes to allow large, non-dissociated fragments to sediment. Afterward, transfer the supernatant to a new tube and spin the cells at 120 times G for seven minutes.
Once pelleted, we suspend the cells in ten milliliters of one percent FCS in HBSS and count them using a Neubauer chamber. Prior to cell staining, transfer one times ten to the sixth intestinal cells to a five milliliters tube to prepare a negative control. To prepare a sample, add one time ten to the sixth of intestinal cells, followed by two milliliters of HBSS with one percent FCS to a new five milliliters tube and centrifuge the suspension at 120 times G, at four degrees Celsius, for five minutes.
After discarding the supernatant, we suspend the cells in 50 microliters of the antibodies solution. Wrap the tube aluminum foil and incubate the cells at four degrees Celsius for 20 minutes. Once the incubation is completed, add two milliliters of one percent FCS in HBSS to the sample and centrifuge it at 120 times G, four degrees Celsius, for five minutes.
Discard the supernatant and resuspend the pellet in 200 microliters of 0.5 micrograms per milliliters propidium iodide solution. After decapitating a mouse embryo, soak it's body in a petri dish, lined with a pre-wet paper towel, and filled with 50 milliliters of one percent FCS in HBSS. Under a stereomicroscope make an incision on the right side of the embryo's chest and carefully open the thorax, avoiding damaging the heart.
Seize the great vessels and pull out the heart connected to the lungs and the thymus. Transfer the organs to a 35 milliliter petri dish filled with two milliliters of one percent FCS in HBSS. Then, isolate the heart from the surrounding organs and connective tissue.
After washing the heart, soak it in one milliliter of pre-warmed enzymatic solution, and using fine forceps and a scalpel, mince the organ into one cubic millimeter pieces under a stereomicroscope. Add an additional one milliliter of pre-warmed enzymatic solution and transfer the fragmented tissue to a capped five milliliter tube. Incubate the sample in a horizontal position at 37 degrees Celsius for 15 minutes.
Once the incubation is completed, homogenize the tissue by repetitive pipetting of the suspension with a P1000 pipette. Then, set the samples, positioned vertically, aside until the fragments of the undigested tissues sediment. Transfer the supernatant to a 50 milliliters tube.
Add two milliliters of ten percent FCS in HBSS and keep the isolated cells on ice. Next, add two milliliters of pre-warmed enzymatic solution to the five milliliters tube containing the undigested tissue sediment, and continue to digest the tissue as shown previously until no remaining tissue is observed. Centrifuge the obtained cell suspension at 120 times G for ten minutes.
Then, discard the supernatant and resuspend the cells in one milliliters of one percent FCS in HBSS, free of calcium and magnesium cations. To stain the cardiac cells, transfer 200 microliters of the cell suspension into wells of a round bottom 96 well plate. Centrifuge the plate at 480 times G for one minute and discard the supernatant.
Next, resuspend the cardiac cells in 100 microliters of the antibody solution. Wrap the plate in aluminum foil and incubate it at four degrees Celsius for 20 minutes. Once the incubation is completed, wash the cardiac cells with 200 microliters of one percent FCS in calcium and magnesium free HBSS, and centrifuge the suspension at 480 times G for one minute.
Then, resuspend the cells in 200 microliters of one percent FCS in HBSS free of calcium and magnesium ion, and transfer the suspension to a five milliliters tube prefilled with 200 microliters of one percent FCS in calcium and magnesium free HBSS. Finally, add 400 microliters of one percent FCS in HBSS free of calcium and magnesium cations, containing 0.5 micrograms per milliliter propidium iodide and filter the cell suspension through a 70 micro nylon mesh. To visualize the cells load the stained sample to a flow cytometry and click preview, then record up to two times ten to the sixth events in the sample by clicking acquire.
In the analysis tab, open the color pallet window and register all studied parameters by adding the fluorochrome along with it's corresponding marker. Make sure to include autofluorescence and viability parameters. In the control window, within tube list, analysis the first single stained sample containing compensation beads.
Then, in the worksheet, click on the polygon design tool and gate the bead population in the FSC SSC plot. Double-click on the gate to create a daughter plot of the gated beads. And then gate positive and negative beads separately.
Verify the selected daughter population by successive gating and elimination of outliers in each positive and negative fraction. Then, upload the negative and positive gates in the un-mixing window. Next, select the unstained cell sample in the tube list and design separate gates for autofluorescent and non-autofluorescent cells.
Once the negative and positive gates are defined for all parameters, including autofluorescence and viability, click on calculate. Then, apply un-mixing to the analyzed samples. To analyze data, gate the cells in a plot of SSC versus FSC and exclude propidium iodide stained dead cells.
Finally, determine gates for all populations of interest. Presented here are results of flow cytometry and spectral cytometry analyses of the cells isolated from fetal heart and stained with anti-TER119, anti-CD45, and anti-Sca-1 antibodies. A subset of autofluorescence cells was detected by two conventional cytometers while in spectral cytometry these cells were not defined as autofluorescent and were comprised in the CD45 TER119 negative population.
The cells identified by conventional flow cytometry as autofluorescent, but not CD31 positive cells were then analyzed for the expression of the transcripts specific to cardiomyocytes. High levels of cardiac troponin and atrial myocytes like train transcripts were found in autofluorescent cells population confirming that a large subset of cardio myocytes is missed in conventional flow cytometry. Once mastered, this technique can be done in approximately eight hours, if it is preformed properly.
While attempting this procedure it's important to remember that all steps shall be performed on ice and in a timely manor to ensure cells remain viable. Following these procedure, intracellular proteins and analyses of additional surface markers can be preform in order to answer questions regarding specific molecular pathways. This technique paved the way for researchers in the field of immunology and developmental or stem cell biology to explore and isolate rare populations from different organs.
After watching this video you should have a good understanding of how to isolate and stain cells from cellular tissues, and how to analyze surface marker expression by spectral flow cytometry that overcomes limitations resulting from cell autofluorescence. Don't forget that working with propidium iodide can be extremely hazardous, and precautions such as the use of PPE should always be taken while performing this procedure.
