TITLE:  Preparation of single-cell suspensions for scMEP mass cytometry analysis 
AUTHORS: Felix Hartmann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Preparation of single cell suspensions to be analyzed for metabolic and phenotypic features by scMEP mass cytometry analysis. This protocols spans 1) optional incubation of cells with small molecules to be able to asses biosynthesis rates of DNA, RNA and protein 2) cisplatin-based live/dead staining 3) PFA-based cell fixation and 4) cryopreservation.</div></div>

[STEPS]
?. [Obtain single-cell suspension]
Prepare single-cell suspension with your established method of choice.Be aware: cell aggregates clog the small tubing of the CyTOF and prevent acquisition.Use of digestive enzymes might be necessary but can lead to epitope loss.Cell numbers depend on the experimental question but preparing 1-3 million cells per sample is recommended.
?. [Analysis of biosynthesis rates (this section is optional)]
Prepare stocks for small molecules:IdU (Recommended stock   in DMSO)BrU (Recommended stock   in PBS)Puromycin (Recommended stock   in ddH2O)Solutions can be gently heated in a   water bath to completely dissolve solute if necessary. Once dissolved, prepare small aliquots and store at   until needed.Reagents: More details:
50 °C
-20 °C
?. [Analysis of biosynthesis rates (this section is optional)]
Prepare mastermix of small molecules in cell-type appropriate medium (can be supplemented with serum or other compounds).   ABCD1CompoundFinal concentrationStock concentrationVolume per sample2IdU100 uM500 mM0.2 uL3BrU2 mM100 mM20 uL4Puromycin5 ug/mL1 mg/mL5 uL5Medium (cell-type specific)1 mL
ABCD1CompoundFinal concentrationStock concentrationVolume per sample2IdU100 uM500 mM0.2 uL3BrU2 mM100 mM20 uL4Puromycin5 ug/mL1 mg/mL5 uL5Medium (cell-type specific)1 mL
?. [Live / dead staining]
Transfer cell suspension to FACS tubes or similar vessel and fill with cell-type appropriate medium (not containing IdU/BrU/puromycin) and centrifuge at  .
Centrifuge: 300 34, 5 min
?. [Live / dead staining]
Pre-dilute Cisplatin-198Pt to  in PBS (Stock ).First time: aliquot Cisplatin stock solution into  aliquots and store at   until needed.Aliquots can be thawed and refrozen multiple times.Discard pre-diluted Cisplatin after use.Reagents:
5 µl
-20 °C
?. [Live / dead staining]
Aspirate supernatant and resuspend cells in  PBS.
1 mL
?. [Live / dead staining]
Add  pre-diluted Cisplatin-198Pt to cells in PBS, vortex and incubate for  at .
1 µl
0 Room temperature
?. [Live / dead staining]
Add  of Cell Staining Medium (CSM: PBS + 0.5% BSA + 0.02% sodium azide) and centrifuge .
[or whatever your vessel allows]
Centrifuge: 300 34, 5 min
?. [Cell fixation]
Aspirate supernatant and loosen cell pellet by flicking tube or vortexing.
?. [Cell fixation]
Dilute 16% PFA to 1.6% in PBS.PFA from Electron Microscopy Sciences has been shown to work well.Opened 16% PFA can be stored airtight and light-protected (e.g. in black falcon tube) for up to two weeks.Diluted 1.6% PFA should be discarded after use.Reagents:
?. [Cell fixation]
Add  of freshly diluted 1.6% PFA to loosened cells, mix well and incubate for  at  to fix cells.
1 mL
0 Room temperature
?. [Cell fixation]
Add  of Cell Staining Medium (CSM) and centrifuge .
[or whatever your vessel allows]
Centrifuge: 600 34, 5 min, 4 10
?. [Freezing]
Aspirate supernatant and resuspend cells in  of CSM + 10% DMSO.
1 mL
?. [Freezing]
Transfer to cryotube and freeze at .
-80 °C
?. [Analysis of biosynthesis rates (this section is optional)]
Resuspend cells in   of mastermix, and incubate at   for .
1 mL
37 °C