Protocols

A platinum based anti-cancer drug called Cisplatin is utilised to identify dead cells much in the way fixable live/dead markers are used in standard flow cytometry. In panels where platinum conjugated antibodies are used we use Rhodium103 as an alternative viability stain. This is added to cells at the start of the labelling procedure and is taken up by cells that are dying or dead within the sample.

Metal conjugated antibodies can be purchased directly from Fluidigm (see the link in the 'Resources' page for links to the catalogue), or conjugated in house. If you are planning a large antibody panel it is likely that you will need to have some custom conjugations done in house; please contact the facility to discuss this further. Nearly all the commercially available antibodies have been validated against peripheral blood mononuclear cells (PBMCs) and are ready to use at a dilution of 1-100. It is advisable however to perform titration of these antibodies. As there is no requirement for single stain controls all the antibodies can be added to the cells in a single cocktail.

Barcoding your samples for CyTOF can save antibody, enable doublet discrimination and standardise data acquisition, depending on the barcoding procedure you use.

• Live cell barcoding - we have a number of barcoding antibodies available within the facility such as CD45 and HLA-A,B,C conjugated to a number of different metals which do not interfere with the lanthanides used in staining panels. These can be used to stain samples in unique combinations before pooling to stain with the mastermix of antibodies. Live cell barcoding allows the procedure to be performed without fixing the cells first.

• Palladium barcoding kit available from Fluidigm. This kit utilises a combination of purified isoforms of palladium (Pd) and allows the labelling of up to 20 samples. These kits can be used in two ways: samples can be labelled prior to antibody staining in order to save on antibody and standardise sample collection during acquisition or samples can be labelled following antibody staining and allows standardisation of data acquisition. Cells must be fixed and permeabilised before using these Pd reagents which may have an effect on antibody binding if performed ahead of staining.

Materials and Buffers

• MaxPar metal conjugated antibody cocktail

• PBS made with MilliQ water and no heavy metal contaminants (no glass or beakers washed with soap) - commercially available PBS is recommended

• Cell Staining Buffer (Fluidigm)

• Fix and Perm Buffer (Fluidigm)

• Fc Block in Cell Staining Buffer (optional)

• Intercalation Solution - 125uM Cell-ID Intercalator-Ir diluted 1-1000 in Fix and Perm Buffer to 125nM solution

• Viability reagent - Cisplatin - 2.5mM Cell-ID Cistplatin diluted 1-1000 in PBS to 2.5uM solution or Rhodium-103 diluted 1-500 in PBS

• 1x eBioscience™ Permeabilization Buffer (ThermoFisher cat no. 00-8333-56)

• Paraformaldehyde solution (16%) diluted to 4% in PBS

• Conical (V) or round bottomed 96 well plate

• Cell freezing solution (90% FBS, 10% DMSO) (required for long term storage)

• (Optional) 'Barcoding tube' for staining each sample with an individual barcode (available from the Flow Cytometry Facility) Downloadable sample labelling form

Protocol for surface staining (adapted from Leipold et al, 2015, Methods Mol Biol. 2015; 1343: 81–95.

Cell Surface staining of cells

1. Prepare a single cell suspension of your sample in the usual, optimised manner resuspended in serum free PBS

2. Place 1-3 million cells into well(s) of a conical (V) or round bottomed 96 well plate

3. Collect the cells by centrifugation at 300g/5 mins

4. Fully resuspend the cell pellet in 50uL of 2.5uM Cisplatin solution

5. Incubate at room temperature for 2 minutes

6. Add 150uL Cell Staining Buffer to quench the reaction

7. Collect the cells by centrifugation at 300g/5 mins

8. Resuspend the pellet in 200uL Cell Staining Buffer to wash

9. Collect the cells by centrifugation at 300g/5 mins

10. Resuspend the cell pellet in 50ul of diluted Fc Blocker solution and incubate for 15 minutes on ice

11. Make Ab cocktail in Cells Staining Buffer buffer to a final volume of 50uL

12. Resuspend cells in 50 µL Ab cocktail for a final staining volume of 100uL

13. Incubate for 30–60 min on ice.

14. Wash 3× in Cells Staining Buffer buffer.

Information

If intracellular staining is being performed continue from step A below, otherwise:

1. Resupend the cell pellet in 100uL of 4% paraformaldehyde solution

2. Incubate for 15 minutes at room temp.

3. Collect the cells by centrifugation at 800g/5 mins*

4. Resuspend pellet in 100uL Intercalation Solution (Maxpar Fix/Perm buffer plus iridium intercalator) and transfer to the appropriate 'barcoding tube', incubate for 1 hour at room temp, or over night in fridge.

5. It is recommended to freeze the samples if they will not be run within 24 hours on the CyTOF -

   • Perform a cell count on the overnight fixed cells

   • centrifuge the cells at 800g/5 mins* and resuspended in cell freezing solution (10% DMSO, 90% FBS) to a final concentration of 10e6 cells/mL

   • Aliquot 200uL volumes into separate freezing tubes and stored at -80degC - they will keep stable for months.

* Note higher speed for centrifugation after fixation to reduce cell loss

Intracellular staining

* All subsequent centrifugation steps should be at 800g/5 mins to minimise the loss of cells following fixation

A. Resuspend in 200 µl of 2 % PFA in PBS

B. Incubate at 4°C overnight.

C. Wash 2× in 1× eBioscience perm buffer

D. Make intracellular staining cocktail in 1× perm. buffer and filter with 0.1 µm spin filter.

E. Incubate on ice for 45 min.

F. Wash 3× in Cells Staining Buffer buffer.

G. Resupend the cell pellet in 100uL of 4% paraformaldehyde solution

H. Incubate for 15 minutes at room temp.

I. Collect the cells by centrifugation at 800g/5 mins*

J. Resuspend pellet in 100uL Intercalation Solution (Maxpar Fix/Perm buffer plus iridium intercalator) and transfer to the appropriate 'barcoding tube', incubate for 1 hour at room temp, or over night in fridge.

K. It is recommended to freeze the samples if they will not be run within 24 hours on the CyTOF -

5. • Perform a cell count on the overnight fixed cells

   • centrifuge the cells at 800g/5 mins* and resuspended in cell freezing solution (10% DMSO, 90% FBS) to a final concentration of 10e6 cells/mL

   • Aliquot 200uL volumes into separate freezing tubes and stored at -80degC - they will keep stable for months.