The overall goal of this sample preparation process is to produce a robust and consistent antibody staining of diverse samples to enable the interrogation of heterogeneous cell populations. This method can help answer questions about cell identity, cell signaling and cell response in a complex heterogeneous cell population. The main advantage of this technique is it allows the measurement of high parameter protein levels at the single cell level.
Demonstrating this procedure will be Aundrietta Duncan, a graduate student from our laboratory. For each single cell suspension sample, re-suspend it at four times 10 to the sixth cells per milliliter in serum free medium, add 500 microliters of freshly prepared 50 MicroMolar Cisplatin per 2/10 into the six cells, and incubate the samples on an orbital shaker with constant mixing at room temperature. After one minute, quench the Cisplatin with an equal volume of wash buffer and centrifuge the cells.
Discard the supernatant and wash the samples two more times by adding 500 microliters of wash buffer, centrifuging the cells, and pouring off the wash buffer. Follow this by two washes in 500 microliters of PBS similarly centrifuging the samples and discarding the supernatant. After the second PBS wash, re-suspend the pellets in 500 microliters of fresh PBS and fix the cells with 500 microliters of 4%PFA.
Pipette the cells to mix. Then place the samples on the orbital shaker with constant mixing for 15 minutes at room temperature. At the end of the incubation, pellet the cells by centrifugation, pour off the supernatant, and follow this by a wash in fresh PBS.
To permeabilize the cells, spin down the cells again and discard the supernatants. Re-suspend the samples in the residual PBS by gentle vortexing, and immediately add one milliliter of 100%methanol per 2/10 10 to the sixth cells with gentle pipetting. Incubate the samples at four degrees for at least one hour, and at the end of the incubation, centrifuge the cells.
Afterwards, pour off the methanol. Then wash the pellets in one milliliter of wash buffer for each milliliter of methanol, followed by two more washes in 500 microliters of fresh wash buffer. Using a 200 microliter pipette set to 50 microliters, transfer the remaining wash buffer from each pellet to a tube of the appropriate corresponding antibody cocktail.
Pipette back up the combined solution without drawing air into the tip by keeping the plunger partially depressed. Then transfer the pipette tip into a 1.5 milliliter microcentrifuge tube containing 500 microliters of wash buffer without releasing the plunger. Release the plunger, drawing up the wash buffer for a total antibody cocktail volume of 50 microliters and label the appropriate corresponding sample with the aspirated antibody cocktail by gentle pipetting.
After one hour at room temperature with constant shaking, wash the samples four times, pouring off the wash buffer each time. To label the cells with iridium, re-suspend the final pellets in 500 microliters of PBS, followed by the addition of 500 microliters of four percent PFA for 15 minutes at room temperature. Next, add 500 microliters of freshly prepared 62.5 nanomolar iridium and PFA to the samples for another 15 minutes of room temperature shaking.
Then, centrifuge the cells, pour off the supernatant, and follow by two washes in 500 microliters of 0.1 percent BSA and filtered deionized water. Analyze the samples by adding normalization beads into the wells of sample plates, pipetting cells into these wells and then performing mass cytometry within 24 hours. Following normalization of the signal intensity, the calibration beads can be removed from the data by gating on the iridium 191 positive bead negative population.
The single cell events can then be gated to remove the debris and cell multiplets using a biaxial plot of iridium 193 negative expression versus the event length. Cisplatin labeling can be used to measure the amount of cell depth. For example, here samples with low and higher quantities of dead cells are shown.
The dead cells can be removed by gating away the platinum 198 positive population, barcoding results in a distinct separation of the samples within the barcoding parameters, allowing the cells to be assigned to their original sample identities. IDU labeling can be used to detect US face cells observed here. Following the initial normalization, de-barcoding and gating, the high dimensional data can be analyzed using the SPADE algorithm to generate a lower dimensional representation of the data that is more amenable to visual interpretation.
Once mastered, this technique can be completed within four to six hours if performed properly. While attempting this procedure, it's important to remember to minimize sample loss while doing the wash step. After watching this video, you should have a good understanding of how to prepare samples for mass cytometry analysis.
Don't forget that working with sodium azide can be hazardous, therefore you should use precautions such as wearing proper PPE when performing this procedure.
