BioMek FX pintool or other compound transfer system

We use the BioMek FX pintool to transfer 100 nl of compound from source plates to cell culture plates. Follow the manufacturer’s directions and safety procedures for using the BioMek FX. The following protocol is used to transfer compounds and then wash the pintool.

• • Stamp compound plate.
• • Stamp cell/assay plate, thereby transferring 100 nl of compound.
• • Wash pintool with solvent and blot on filter paper three times using the following sequence of HPLC grade solvents.
  • ○ DMSO
  • ○ Isopropyl alcohol
  • ○ Methanol
• • After blotting the pintool with the last solvent wash of methanol, dry with fan. Replace filter paper frequently.

Any method of transferring compounds to a cell plate with sufficient throughput and precision may be used.

Alternatives: Labcyte Echo with following modification to the protocol: compounds are spotted onto empty cell culture plates prior to adding cells.

BioTek EL-405 or other plate washer

This plate washer is used to quickly and consistently aspirate supernatant from centrifuged plates in order to wash cells. The plate washer should be set to leave a thin layer of supernatant covering the bottom of the well to minimize cell loss and prevent cells from drying out between steps in the protocol. This will need to be optimized for individual plate washers and 384 well plates. We have set our EL-405 plate washer to aspirate to a height of 3.81 mm from the surface of the plate carrier, which leaves about 7 to 9 μl in the wells. It is important to clean plate washers thoroughly and frequently with water and/or ethanol to prevent clogs.

If a plate washer is unavailable, supernatants may be removed by flicking plates upside down over an appropriate receptacle. This should only be done after cells have been pelleted and only a single, quick, sure flick should be performed with each plate, to prevent cell loss. However, this flick method may lead to inconsistent volume left in wells, increased cell loss, and/or inconsistent results across different investigators.

CRITICAL: Be sure to follow best safety practices, institutional policies, and local regulations when handling or disposing of hazardous chemicals or biohazardous waste.

BioTek MicroFlo Reagent Dispenser

A peristaltic pump-based reagent dispenser should be used to rapidly and precisely dispense reagents to plates.

Alternatives: Thermo Fisher Multidrop Combi

CyAn ADP flow cytometer and HyperCyt Autosampler

We used a Beckman Coulter ADP flow cytometer and a Hypercyt Autosampler for this screen. Any flow cytometer that can analyze 384 well plates may serve as an alternative.

Alternatives: Sartorius iQue 3 or the BioRad ZE5 systems.

Titrate IFN-γ Activity

Timing: 3–4 days

IFN-γ should be titrated to determine the activity of a given lot of IFN-γ and THP-1 cells, and to determine the effective concentration at which 20% (EC20) and 50% (EC50) maximal PD-L1 expression is achieved. We recommend performing a dose response curve with at least 12 concentrations of IFN-γ and at least 10 replicate wells for each concentration. This can be adapted for other stimuli applied to cells.

• 4.

  Centrifuge THP-1 cells at 300 × g for 5 min. Resuspend cells in fresh media at 1.25 million viable cells per mL.

• 5.

  Distribute 40 μl of cell suspension per well to a 384 well plate using a reagent dispenser (e.g., the BioTek MicroFlo) or multichannel pipette.

• 6.

  Prepare dilution of IFN-γ.

  • a.

    IFN-γ is prepared at 5X of its desired final concentration.

  • b.

    A serial dilution should be performed. Start with IFN-γ diluted in THP-1 media at 10 μg/mL. Dilute by a factor of 2 to generate at least 12 concentrations (the final dilution should be about 4.9 ng/mL IFN-γ). Table 1 shows directions for performing an example serial dilution.

    Table 1

    Example serial dilution of IFN-γ

    Dilution	IFN-γ (μL)	THP-1 media (μL)	[IFN-γ] (ng/mL) – 5X solution	[IFN-γ] (ng/mL) – Final concentration
    1	4 (from 1 mg/mL stock)	396	10000	2000
    2	200 (from dilution 1)	200	5000	1000
    3	200 (from dilution 2)	200	2500	500
    4	200 (from dilution 3)	200	1250	250
    5	200 (from dilution 4)	200	625	125
    6	200 (from dilution 5)	200	312.5	62.5
    7	200 (from dilution 6)	200	156.25	31.25
    8	200 (from dilution 7)	200	78.13	15.63
    9	200 (from dilution 8)	200	39.06	7.81
    10	200 (from dilution 9)	200	19.53	3.91
    11	200 (from dilution 10)	200	9.77	1.95
    12	200 (from dilution 11)	200	4.88	0.98

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• 7.

  Transfer diluted IFN-γ to cell wells using a multichannel pipette. Transfer 10 μl per well, for a final well volume of 50 μl, and 50,000 cells per well. Each dilution should have at least 10 replicate wells.

• 8.

  Add 10 μl of media without IFN-γ to a set of 10 replicate wells to serve as an unstimulated control.

• 9.

  Incubate cells in a standard humidified 37°C 5% CO2 incubator for three days. Proceed with staining and flow cytometry analysis as described below beginning with step 21.

• 10.

  Graph the median fluorescence intensity of PD-L1 vs IFN-γ concentration in log. Generate a dose response curve and calculate the EC20 and EC50 for IFN-γ using GraphPad Prism or a similar program. In GraphPad Prism, the analysis method “[Agonist] vs. response – Find ECanything” can be used to calculate the EC20.

Note: We recommend performing the screen with cells treated with the calculated EC20 of IFN-γ because we have observed that this relatively low level of stimulation allows for identification of both activators and inhibitors of PD-L1 expression. It also allows for the identification of compounds which might only have a moderate effect on PD-L1 expression but still present interesting avenues for future research. Treatment with higher levels of IFN-γ might eclipse the effect of compounds which activate PD-L1 expression, because expression may “max out” at a certain point. However, if the investigator is only interested in inhibitors of PD-L1 expression (or a different protein for which this protocol might be adapted), it may be more relevant to treat cells with a higher level of cytokine stimulation, such as the EC50 or EC80. Calculations can easily be adjusted accordingly with the “Find ECanything” function in GraphPad prism.

Stimulate cells with IFN-γ and treat with compounds

Timing: 1 day

The screen is initiated by stimulating THP-1 cells and treating with screening compounds. This assay is designed to detect compounds which decrease or increase PD-L1 expression.

• 11.

  If compound source plates are frozen, remove them from the freezer at least 2 h before the planned transfer of compounds to allow ample time for them to equilibrate to approximately 20°C.

  • a.

    Ensure plates are well sealed with an aluminum seal when they are removed from and returned to cold storage.

  • b.

    After compound source plates have equilibrated to approximately 20°C, mix them well by vortexing at a high speed with the HT-91002 microplate orbital shaker in order to ensure the compounds are well dissolved following thawing. Plates should be vortexed for at least 1 min, until compounds are completely dissolved.

  • c.

    After the compound plates have been mixed, centrifuge them at approximately 20–25°C (a refrigerated centrifuge will cause DMSO in the source plates to freeze) for 10 min at 1000 × g in order to ensure no compound remains on the aluminum foil seals. This is critical to ensure there is no cross contamination between wells.

• 12.

  Mix THP-1 cells well. Count THP-1 cells with a hemocytometer and 0.4% Trypan Blue.

  • a.

    Calculate the number of cells needed for the screen. You will need 21 million viable cells per full 384 well plate.

• 13.

  Measure out sufficient THP-1 cells for the screen. Centrifuge cells for 5 min at 300 × g.

• 14.

  Aspirate the supernatant and resuspend in fresh culture media at a concentration of 1 million cells per mL (21 mL per screening plate).

  • a.

    Pipette 1 mL of resuspended cells per plate into a separate centrifuge tube. These cells will be kept as unstimulated controls.

• 15.

  Add IFN-γ to the main tube of cells to a concentration equivalent to the EC20 of activation as determined in the previous IFN-γ titration section. In our hands, this is frequently around 50 ng/mL. Mix well by inverting the tube or pipetting up and down 5 to 10 times.

• 16.

  Distribute IFN-γ stimulated cells to the first 23 columns of the black 384 well assay plates, 50 μl per well (50,000 cells per well). Distribute the unstimulated cells to column 24, also at 50 μl per well.

Note: The placement of control wells within the plate can be altered as the investigator prefers.

CRITICAL: If a multidrop or other reagent dispenser is being used to distribute cells, about 5 mL of extra cell suspension should be prepared, at the same concentration of cells and IFN-γ. This is to account for the dead volume of the reagent dispenser tubing and loss of cells during priming of the tubing.

• 17.

  Carefully label or barcode cell plates so they are clearly matched to compound source plates.

• 18.

  Remove the aluminum foil seal from a compound plate. Use the BioMek FX to transfer 100 nl of compound from the compound source plate to the cell plate. Seal compound plate with a new aluminum foil seal.

• 19.

  Repeat step 18 for each assay plate and compound source plate pair.

• 20.

  Incubate cells at 37°C 5% CO₂ for 3 days.

Stain cells for flow cytometry analysis

Timing: 1 day

After cells have been incubated for 3 days with IFN-γ and compounds, they can be stained for flow cytometry analysis. All centrifuge steps are performed for 5 min at 300 × g, at 4°C, unless otherwise noted. All reagents should be kept at 4°C or on ice unless otherwise noted.

Note: This protocol is intended to detect cell-surface expressed PD-L1 and as such does not require a permeabilization step. The detection of intracellular targets will require membrane permeabilization with reagents such as saponin or Triton X-100 and likely will require further optimization. Permeabilization should only be performed on cells which have already been fixed.

• 21.

  Remove vials of Fixable Viability Dye (Thermo fisher Scientific) 660 from the freezer to allow them to thaw and equilibrate to approximately 20°C. Do not place vials on ice, as this will prevent the DMSO solvent from thawing.

• 22.

  Centrifuge cell plates.

• 23.

  Aspirate supernatant with the plate washer, leaving a thin layer of supernatant covering the pelleted cells.

• 24.

  Wash cells by dispensing 80 μl cold (around 4°C) DPBS without calcium or magnesium per well, then centrifuge and aspirate supernatant. From this point onwards, the plates should be kept on ice or refrigerated as much as possible. (Troubleshooting 1)

Note: Cells are initially washed with DPBS, not FACS buffer, as protein-free DPBS reduces the background staining of the viability dye.

Note: Fixable viability dyes are available in several colors. We chose this color (660, detected in the APC channel on most flow cytometers) because it is excited by the red laser on our Cyan ADP flow cytometer, whereas the fluorophore for our PD-L1 antibody is excited by our blue laser. This minimizes the possibility of spectral overlap between the two fluorophores. However, other color viability dyes are acceptable and should be selected with consideration of your chosen flow cytometer’s optical configuration.

• 25.

  Repeat step 24.

• 26.

  While the cells are centrifuging, mix viability dye concentrate by vortexing. Briefly spin down the tube with a benchtop microcentrifuge. Ensure the dye has completely thawed. Dilute the viability dye 1:1000 in cold PBS. Prepare 16 mL per plate, plus an extra 5 mL to account for the dead volume of the reagent dispenser tubing. Viability dye dilutions should always be prepared fresh.

• 27.

  After the second wash cycle with DPBS (DPBS has been dispensed, cells have been centrifuged, and supernatant has been aspirated), dispense 40 μl diluted viability dye per well. Vortex each plate without a lid with the HT-91002 microplate orbital shaker at speed setting 700 (arbitrary units) for about 5 s to resuspend the cells. From this point onwards, plates should be protected from light. Incubate plates at 4°C, with gentle rocking or orbital shaking, in the dark, for 30 min.

Note: If you are using the HT-91002 microplate orbital shaker, or an alternative shaker, for the first time, gradually increase the speed to find a setting where the plate can be shaken vigorously enough to resuspend the cells without spilling the contents of the plate. The investigator can ensure that cells are being thoroughly resuspended by looking at the plate with a standard light microscope.

Optional: If you have not used the viability dye before, or if you are performing a flow cytometry assay with more than two colors, you can heat kill cells to create a positive control for viability dye staining. Consult the manufacturer’s instructions for more information.

• 28.

  Following the incubation, wash cells by dispensing 40 μl FACS buffer per well. Centrifuge.

• 29.

  Aspirate the supernatant with the plate washer. Wash cells again by dispensing 80 μl per well FACS buffer. Centrifuge.

• 30.

  While cells are centrifuging, dilute Human FcR Blocking Reagent 1:20 in FACS buffer. 15 μl per well or about 6 mL per plate is required. FcR Blocking Reagent dilutions should always be prepared fresh.

Note: The FcR Blocking Reagent prevents non-specific binding of antibodies to Fc receptors. Fc receptors are expressed on a variety of immune cells.

• 31.

  After plates have been centrifuged, aspirate supernatant and dispense 15 μl per well diluted Human FcR Blocking Reagent. Vortex plates without lids using the plate shaker, at speed setting 700 for 5 to 10 s each.

• 32.

  Incubate plates at 4°C, with gentle rocking or orbital shaking, in the dark, for 10 min.

• 33.

  During this incubation, prepare antibody dilutions. The PE conjugated antibody against human PD-L1, should be diluted 1:60 in FACS buffer (for an ultimate dilution of approximately 1:150). Enough volume for 15 μl per well (about 6 mL per plate with dead volume) is required. Antibody dilutions should always be prepared fresh.

Note: We have used an anti-PD-L1 antibody conjugated to phycoerythrin (PE) because PE is one of the brightest fluorophores available for flow cytometry. Brighter fluorophores may make it easier to distinguish antibody staining signal from background fluorescence. However, any other fluorophore which has minimal spectral overlap with the viability dye should be acceptable. Always confirm that a given fluorophore is compatible with your flow cytometer before selecting it.

• 34.

  Following the 10 min incubation, without centrifuging the plates, dispense 15 μl per well of diluted antibody. We recommend leaving some wells unstained as a control. Vortex plates without lids using the plate shaker, at speed setting 700 for 5 to 10 s each.

Note: The FcR Blocking Reagent does not need to be removed before adding the antibody solution.

• 35.

  Incubate plates at 4°C, with gentle rocking or orbital shaking, in the dark, for 45 min.

• 36.

  Following the 45 min incubation, dispense 40 μl per well FACS buffer. Centrifuge plates.

• 37.

  Aspirate supernatant, dispense 80 μl per well FACS buffer. Centrifuge plates.

• 38.

  Aspirate supernatant, vortex plates without lids using the plate shaker, at speed setting 700 for 5 to 10 s each.

• 39.

  Dispense 40 μl per well 4% paraformaldehyde (PFA) (Electron microscopy Services), diluted from the 32% stock in DPBS. 4% PFA solution should be prepared fresh.

CRITICAL: PFA is hazardous and should be handled in a fume hood using appropriate PPE and disposed of properly.

• 40.

  Incubate for 15 min at approximately 20°C, with gentle rocking or orbital shaking, with plates protected from light.

• 41.

  Dispense 40 μl per well FACS buffer. Centrifuge plates, aspirate supernatant, and vortex plates to resuspend cells. PFA waste should be collected and handled as hazardous chemical waste and disposed of according to local regulations and institutional policies.

• 42.

  Dispense 50 μl per well FACS buffer. Vortex plates again to ensure cells are resuspended and well mixed. The plates are now ready to analyze.

Pause point: Stained, fixed plates can be stored at 4°C, in the dark before flow cytometry analysis for 1 week.

Potential solution

This may indicate that too high concentrations of IFN-γ are being used. Lower the amount of IFN-γ. If the problem persists, the cells can be washed with PBS supplemented with 1 mM EDTA to help loosen any adhesion to plates. Ultra-low attachment 384 well plates can also be employed.

Problem 2

Low number of events per well during flow cytometry analysis.

Potential solution

This may be caused by cells which have settled in the wells or remain partially pelleted. Vortex plates thoroughly and increase the frequency of inter-well shaking during flow cytometry analysis from every 12 wells to every 6 wells. The investigator can also decrease the volume of FACS buffer in the final resuspension of cells.

Additionally, investigators can try increasing the sample time for each well, but this tends not to work as well as decreasing the volume of the cell suspension.

Problem 3

THP-1 cells do not express PD-L1 consistently or at high levels following IFN-γ stimulation.

Potential solution

THP-1 cells which have been in culture for too long (“high passage”) or have been cultured at too high a density may have inconsistent response to IFN-γ. Thaw a new stock of low passage THP-1 cells. Also, consider redoing the IFN-γ titration to ensure that stocks have not lost potency. This is especially pertinent if a new batch of IFN-γ has been purchased or if the existing batch has been in storage for more than 1 year.

Problem 4

“Edge effects” or other positional bias observed.

Potential solution

In any high throughput screening assay, there is a risk of bias occurring due to the position of wells within a plate. This is commonly seen in the form of “edge effects”, where the edge wells of a plate will provide different responses due to differences in media evaporation, temperature changes or other factors. This can be mitigated by not using the two rows or columns on the edges of each side of a 384 well plate, and instead filling those wells with sterile PBS. Additionally, allowing the plate to incubate at approximately 20°C for 1 h after adding cells to the plate can reduce edge effects (Lundholt et al., 2003). Plates can also be sealed with gas permeable seals. Furthermore, investigators can use statistical methods to analyze data which account for positional effects, such as the B-score metric (Malo et al., 2010).

Problem 5

High background fluorescent signal from negative control cells (without IFN-γ) or low signal from positive control cells (with IFN-γ and DMSO).

Materials availability

This study did not produce unique reagents.

The authors thank Emily N. Chin and Philipp N. Sander for insightful discussions.