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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Staining of single-cell suspensions for scMEP analysis by mass cytometry (CyTOF). This protocol includes 1) surface staining for 30min at RT, 2) PFA-fixation for 10min at RT, 3) MeOH-based permeabilization for 10min on ice 4) intracellular staining for 1h at RT and 5) DNA intercalation.</div></div>

[STEPS]
?. [Obtain single cell suspension]
Prepare single cell suspensions in FACS tubes as described in the following protocol:
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?. [Barcoding (optional)]
Perform palladium-based barcoding as described in the following protocol:(still needs to be written)
?. [Surface staining]
Prepare surface antibody mastermix. Note: Antibodies were titrated for 3 million cells, if more cells are to be used, scale antibody amounts accordingly (i.e. use 2x antibodies for up to 6 million cells). Combine all surface antibodies and supplement volume to a total of  with Cell Staining Medium (CSM: PBS + 0.5% BSA + 0.02% sodium azide). If antibody volume succeeds  , no CSM addition is needed.
50 µl
50 µl
?. [Surface staining]
Add antibody mastermix to cells and mix carefully by pipetting up and down, followed by a gentle vortex. Incubate for   at  .
0 Room temperature
?. [Surface staining]
Add  of CSM, mix carefully and centrifuge  .
2 mL
Centrifuge: 600 34, 5 min
?. [Surface staining]
Aspirate supernatant and dissolve pellet by flicking tube or vortexing.
?. [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:
?. [Fixation]
Add  of freshly diluted 1.6% PFA to loosened cells, mix well and incubate for  at   to fix antibodies onto cells. Also complete this section when working with pre-fixed cells.
1 mL
0 Room temperature
?. [Fixation]
Add  of Cell Staining Medium (CSM) and centrifuge . Aspirate supernatant and loosen cell pellet by flicking and vortexing.
[or whatever your vessel allows]
Centrifuge: 600 34, 5 min, 4 10
?. [Permeabilization]
Note: MeOH based permeabilization has been found to work well with intracellular metabolic antibodies although most are suspected to work with saponin-based approaches as well.While vortexing cells, drop-wise add  of pre-cooled ( ) MeOH and immmediately transfer cells  to incubate for  .
1 mL
4 °C
on ice
?. [Permeabilization]
Following incubation, add  of CSM and centrifuge  . Aspirate supernatant and loosen cell pellet.
[or whatever your vessel holds]
Centrifuge: 600 34, 5 min, 4 10
?. [Permeabilization]
Repeat washing by adding  of CSM and centrifuge  . Aspirate supernatant and loosen cell pellet.
[or whatever your vessel holds]
Centrifuge: 600 34, 5 min
?. [Intracellular staining]
Prepare intracellular antibody mastermix. Note: Antibodies were titrated for 3 million cells, if more cells are to be used, scale antibody amounts accordingly (i.e. use 2x antibodies for up to 6 million cells). Combine all intracellular antibodies and supplement volume to a total of  with CSM. If antibody volume succeeds  , no CSM addition is needed.
50 µl
50 µl
?. [Intracellular staining]
Add antibody mastermix to cells and mix carefully by pipetting up and down, followed by a gentle vortex. Incubate for   at  . Flick tube carefully after approximately  to ensure proper mixing of antibodies and cells.
0 Room temperature
?. [Intracellular staining]
Add  of CSM, mix carefully and centrifuge  . Aspirate supernatant and loosen pellet.
2 mL
Centrifuge: 600 34, 5 min
?. [DNA intercalation]
Prepare intercalation solution: 1.6% PFA in PBS +   of Intercalator-Rh.Note: Iridium-based DNA intercalators can be used alternatively, however they do tend to spill into adjacent platinum channels which are often used here for live/dead discrimination as well as for antibody channels. Rhodium DNA intercalation is compatible with palladium-based barcoding.When using for the first time, aliquot Rh-intercalator and store at    until useRh-intercalator can be thawed and re-frozen multiple timesWhen using for the first time, aliquot Rh-intercalator and store at   until use
-20 °C
?. [DNA intercalation]
Add  of intercalation solution for each 10 million cells and incubate either  at  or  at  .
1 mL
0 Room temperature
4 °C
?. [Acquisition]
First CSM wash step: Add  of CSM to the cells and centrifuge  . Aspirate supernatant and loosen cell pellet.
[or whatever the vessel holds]
Centrifuge: 600 34, 5 min
?. [Acquisition]
Second ddH2O wash step: Add  of ddH2O to the cells and centrifuge  . Aspirate supernatant and loosen cell pellet.
[or whatever the vessel holds]
Centrifuge: 600 34, 5 min
?. [Acquisition]
Third ddH20 wash step: Add  of ddH2O to the cells and centrifuge  . Aspirate supernatant and loosen cell pellet.
[or whatever the vessel holds]
Centrifuge: 600 34, 5 min
?. [Acquisition]
Dilute EQ beads 1:10 in ddH2O and add  of diluted beads per 1 million cells.
1 mL
?. [Acquisition]
Filter cells through cell strainer and acquire on CyTOF