TITLE: Processing of fixed spheroids for TOBis barcoding, enzyme-free dissociation and antibody staining for CyTOF
AUTHORS: Ralitsa R Madsen

[DESCRIPTION]
This protocol is an adaptation and extension of the original work on single-cell signalling in organoids by the Tape Lab at UCL. In this adaptation, I have optimised a new approach for non-enzymatic single-cell dissociation of fixed, scaffold-free spheroids. The adapted protocol is also shorter and uses less material throughout. 

Its development would not have been possible without the initial help provided by the Tape Lab in 2021-2022, particularly by Jahangir Sufi. For their original protocol, please refer to the following publication: Sufi, J., Qin, X., Rodriguez, F.C. et al. Multiplexed single-cell analysis of organoid signaling networks. Nat Protoc 16, 4897–4918 (2021). https://doi.org/10.1038/s41596-021-00603-4

[STEPS]
SECTION: Spheroid collection
1. Generate spheroids in Elplasia plates (96-well or 24-well plate format) according to the following protocol: dx.doi.org/10.17504/protocols.io.3byl4bnrrvo5/v1

SECTION: Spheroid collection
2. At the end of your experiment, add 16 % formaldehyde straight to the culture medium for a final dilution to 4 % (e.g., for 96-well plate - add  50 µL 16 % formaldehyde to 150 µL  medium solution; for 24-well plate - add 500 µL 16 % formaldehyde to 1.5 mL medium).

NB: I recommend using an electronic multichannel pipette at low dispensing speed. 

SECTION: Spheroid collection
3. Leave the plate on ice and covered with foil for 60 min

SECTION: Spheroid collection
4. Using non-filter aspiration tips, remove the fix solution and add 200 µL PBS to each well in a 96-well Elplasia plate or 2 mL to wells in a 24-well plate.

NB: if working with 24-well Elplasia plates, to avoid aspirating the spheroids, I recommend using non-filter, gel-loading tips. 
 
SECTION: Spheroid collection
4.1. It is possible to parafilm the plate at this point and store in the fridge for further processing. I have tested this for plates stored up to 4 months with successful results. Longer storage periods may also work but have not been tested by me or members of my lab. 
SECTION: TOBis barcoding
5. Depending on the number of samples to be multiplexed, thaw either the 35-plex or 126-plex barcodes at room temperature just before use. Mix and short-centrifuge to pull contents to the bottom of the tubes.
SECTION: TOBis barcoding
6. Remove the old PBS from the wells, and replenish with 72 µL (96-well) or 270 µL (24-well) MaxPar PBS. 

SECTION: TOBis barcoding
7. Add 8 µL (96-well) or 30 µL (24-well) of each barcode solution to the respective wells.

NB: make sure to have a pre-printed code assignment sheet as per the attached example. 

 
 

SECTION: TOBis barcoding
8. Parafilm the plate and leave on a rocker 60 min at 4 °C

SECTION: Excess barcode neutralisation with glutathione (GSH)
9. Once the incubation period is complete and just before you need it, make up ‘x’ ml of 2 mM Glutathione (GSH) in MaxPar CSB (Cell Staining Buffer), for example: 49.2 mg of GSH to 80 ml of CSB, dissolve at room temperature by shaking for 10-20 minutes.

NB: adjust the amount depending on the total number of plates being processed and total volume that will be needed
SECTION: Excess barcode neutralisation with glutathione (GSH)
10. Remove the barcode solution from each well, then wash with 200 µL (for 96-well plate) or 1 mL (for 24-well plate) of 2 mM GSH in CSB. Incubate for 10 minutes, then repeat the wash/incubation another 2 times for a total of 3 times.

NB: continue to use gel tips for aspiration of solutions in 24-well plate. 

SECTION: Excess barcode neutralisation with glutathione (GSH)
11. Next, wash with 200 µL (for 96-well plate) or 1 mL (for 24-well plate) of PBS. Incubate for 10 minutes, then repeat the wash/incubation once more for a total of 2 times.

NB: continue to use gel tips for aspiration of solutions in 24-well plate. 
SECTION: TissueGrinder single-cell dissociation of barcoded spheroids
12. Pre-coat a TissueGrinder tube with 10 mL CSB, making sure to invert the tube to cover all parts, including the blades. Reuse the coating solution to coat an additional 50 ml Falcon for spheroid collection. 

NB: for up to 126 conditions with unique barcoding, a single Tissue Grinder tube is needed for dissociation. Increase as needed for multiple independent experiments. 
SECTION: TissueGrinder single-cell dissociation of barcoded spheroids
13. Using the final PBS on the spheroids and CSB-precoated tip (P200 for 96-well; P1000 for 24-well), pipette up-and-down forcefully and transfer the spheroid suspension to a collection boat, then to the CSB pre-coated 50 ml Falcon using a CSB pre-coated 10 ml stripette. Add 5 mL PBS to the boat, and use to perform one final wash of each well (multichannel), pipetting across all wells in turn and collecting into the final suspension. 

SECTION: TissueGrinder single-cell dissociation of barcoded spheroids
14. Spin the spheroids down at 800 x g, 5 min , (this is very important, otherwise the spheroids will dislodge when the centrifuge stops).

NB: if the spheroids have not pelleted properly, remove as much of the supernatant as possible without touching the spheroids, then use CSB pre-coated tips to transfer the spheroids to CSB pre-coated 1.5 ml Eppendorf tubes (use several if necessary) and repeat the centrifugation using a  benchtop centrifuge at 800 x g, 3 min

Alternative to spin: you can also let the spheroids settle under gravity. Leave unperturbed for 5 minutes. 
SECTION: TissueGrinder single-cell dissociation of barcoded spheroids
15. Remove as much of the supernatant as possible, then use a CSB pre-coated tip to resuspend the spheroids in MaxPar PBS supplemented with 2 mM EDTA for a final volume of approximately 800 µL (taking into account the volume taken up by the spheroids).
SECTION: TissueGrinder single-cell dissociation of barcoded spheroids
16. Keeping the TissueGrinder tube lid inverted on a flat surface and using a CSB pre-coated tip, transfer the spheroid suspension into the lid, fitting it between the blades, then screw on the tube in inverted orientation. 
SECTION: TissueGrinder single-cell dissociation of barcoded spheroids
17. Proceed with dissociation using the standard TissueGrinder protocol "Harsh" (3 min). 

SECTION: TissueGrinder single-cell dissociation of barcoded spheroids
18. Spin down the TissueGrinder tube with single cells at 800 x g, 5 min .
SECTION: TissueGrinder single-cell dissociation of barcoded spheroids
19. Without removing the supernatant, wash the blades/strainer with another 2 mL CSB to retrieve any cells that may have been left behind. Repeat centrifugation at 800 x g, 5 min 

SECTION: TissueGrinder single-cell dissociation of barcoded spheroids
20. Gently, remove the supernatant and use a CSB pre-coated tip to resuspend the pellet in 1 mL CSB, followed by transfer of the suspension to a CSB pre-coated 1.5 ml Eppendorf tube. 

SECTION: TissueGrinder single-cell dissociation of barcoded spheroids
21. Count the cells using a haemocytometer (I have found that this is more reliable for fixed, dissociated cells compared to automated counters; if the suspension is dense, dilute a small aliquot up to 1:10 in PBS for counting).

Calculate how many cells will be needed for staining (up to ~4 million cells can be used per staining round). 

NB: the dissociated cells can be stored in CSB for up to 4 weeks at 4 °C.

SECTION: Extracellular antibody staining
22. Prepare the extracellular antibody staining cocktail. This requires individual antibody optimisation and titration. An example is provided below, with emphasis on the use of non-overlapping metals (also, checks against intracellular targets). The final volume should be 50 µL, with CSB as the diluent.

 

SECTION: Extracellular antibody staining
23. Using a CSB pre-coated tip, take forward the required amount of dissociated cells for staining in 1.5 ml Eppendorf tube and centrifuge in a benchtop centrifuge at800 x g, 3 min


SECTION: Extracellular antibody staining
24. Discard the supernatant and proceed with addition of the 50 µL extracellular antibody cocktail.
SECTION: Extracellular antibody staining
25. Incubate for 30 min with continuous vortexing at low speed (use tape to secure the tube in an upright position on the vortex). 

SECTION: Extracellular antibody staining
26. Once the previous step has completed, add 1 mL CSB to the suspension and centrifuge in a benchtop centrifuge at1500 x g, 5 min

Discard the supernatant.
SECTION: Permeabilisation
27. Resuspend the cell pellet in 200 µL of 0.1 % Triton X-100 (diluted in MaxPar PBS), gently vortex, and incubate at room temperature for 30 min under low-speed vortexing (use tape to secure the tube in an upright position on the vortex). 

SECTION: Permeabilisation
27.1. In the meantime, prepare the intracellular antibody staining cocktail. This requires individual antibody optimisation and titration. An example is provided below, with emphasis on the use of non-overlapping metals (also, checks against intracellular targets). The final volume should be 50 µL, with CSB as the diluent.
 
SECTION: Permeabilisation
28. Once the previous step has completed, add 1 mL CSB to the suspension and centrifuge in a benchtop centrifuge at1500 x g, 5 min

SECTION: Permeabilisation
29. Discard the supernatant and place the cells on ice °C for 1 min.

SECTION: Permeabilisation
30. Resuspend the cells in 200 µL ice-cold 50% methanol (diluted in MaxPar PBS and stored at -20 °C until use), gently vortex and incubate for 10 min at °C on ice.

SECTION: Permeabilisation
31. Once the previous step has completed, add 1 mL CSB to the suspension and centrifuge in a benchtop centrifuge at1500 x g, 5 min

SECTION: Permeabilisation
32. Discard the supernatant and repeat the previous step, taking care to discard all the supernatant afterwards (NB: residual supernatant can affect the subsequent intracellular antibody staining).
SECTION: Intracellular antibody staining
33. Add the 50 µL intracellular antibody cocktail to the cell pellet.
SECTION: Intracellular antibody staining
34. Incubate for 30 min with continuous vortexing at low speed (use tape to secure the tube in an upright position on the vortex)
SECTION: Intracellular antibody staining
35. Once the previous step has completed, add 1 mL, CSB to the suspension and centrifuge in a benchtop centrifuge at1500 x g, 5 min

SECTION: Intracellular antibody staining
36. Discard the supernatant and repeat the previous step. Remove the supernatant.
SECTION: Antibody fixation &amp; Intercalation
37. Gently vortex the cell pellet, then add 200 µL fresh 1.6 % fresh formaldehyde solution, vortex gently and incubate for 10 min at room temperature.

NB: including this fix step increases debar coding efficiency and stained cells can be kept in the fridge for up to 2 weeks and no longer.
SECTION: Antibody fixation &amp; Intercalation
38. Once the previous step has completed, add 1 mL, CSB to the suspension and centrifuge in a benchtop centrifuge at 2000 x g, 5 min

NB: the higher centrifugation speed is needed at this point as pelleting is not as efficient after the previous step. 
SECTION: Intracellular antibody staining
36.1. During the centrifugation, prepare a fresh 1.6% formaldehyde solution with MaxPar PBS as the diluent. 
SECTION: Antibody fixation &amp; Intercalation
38.1. During the centrifugation, dilute 1 µL Intercalator (191-Ir & 193-Ir) in 1 mL Fix & Perm Buffer.

SECTION: Antibody fixation &amp; Intercalation
39. Discard the supernatant and resuspend the cell pellet in 500 µL of diluted Intercalator, gently vortex and incubate for 60 min at room temperature or leave overnight in the fridge at 4C (close lid tightly!).

NB: cells can be left at 4C in the Intercalator for up to 4320 min, however, staining intensity may decrease.

SECTION: Prepare for acquisition
40. Without removing the supernatant, add 1 mL CSB to the cell-Intercalator suspension and centrifuge in a benchtop centrifuge at 2000 x g, 5 min

SECTION: Prepare for acquisition
41. Remove the supernatant, taking care not to dislodge the pellet (repeat centrifugation if that is the case), then add 2x 500 µL MaxPar CAS+ supplemented with EDTA for 2 mM final concentration (e.g. 4 µL of 0.5 M EDTA to 1 mL CAS+ solution); in rounds of 2x 500 µL transfer the solution to a FACS tube with a 0.35 µm cell strainer lid.

SECTION: Prepare for acquisition
42. Thoroughly vortex the EQ6 Beads, then add 1:5 beads to cell suspension solution (e.g., 250 µL EQ6 beads to 1 mL cell suspension solution).

SECTION: Prepare for acquisition
43. Proceed with sample acquisition on the XT, taking care to specify the correct combination of metals in the template.