Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Drug preparation
• Procedure: Splenocyte preparation and drug treatment
• Procedure: Splenocyte staining with fluorescent antibodies
• Procedure: Flow cytometry
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected *
1	Spleen harvested and cell suspension prepared	Dissecting kit, frosted glass slides	-
2	Splenocytes plated	96-well plate	-
3	Splenocytes treated with one of four anti-cancer drugs at various doses for 4h	Incubator	-
4	Spenocytes washed, labeled for immune cells, washed, and fixed	Incubator, centrifuge	-
5	Samples analyzed by flow cytometry	FACSCanto II flow cytometer	Viable B cells, T cells, and monocytes

*Supplementary data are available for this project. See below.

Equipment and supplies

• Frosted glass microscope slides (Thermo Fisher Scientific, Pittsburgh PA)
• Tissue culture dishes (TPP, Trasadingen Switzerland)
• 5 mL polystyrene round-bottom tubes (BD Biosciences, San Jose CA)
• Centrifuges
• 96-well round-bottom plates (Globe Scientific Inc, Paramus NJ
• Tissue culture incubator (5% CO₂)
• FACSCanto II flow cytometer (BD Biosciences, San Jose CA)
• FlowJo version X analysis software (TreeStar, Ashland OR)

Reagents and solutions

• Phosphate-buffered saline (PBS)
• Fetal bovine serum (FBS; Cellgro, Manassas VA)
• Ammonium-chloride-potassium lysing buffer (Gibco, Grand Island NY)
• RPMI-1640 buffer (Cellgro, Manassas VA )
• 2-mercaptoethanol (MP Biomedicals, Santa Ana CA)
• Sodium pyruvate (Cellgro, Manassas VA)
• Nonessential amino acids (Cellgro, Manassas VA)
• Penicillin G/streptomycin solution (Cellgro, Manassas VA)
• BEZ235 (Novartis, Inc.)
• Doxorubicin (Sigma-Aldrich, Milwaukee WI)
• Idarubicin (Sigma-Aldrich, Milwaukee WI)
• Selumetinib (ChemieTek, Indianapolis IN)
• Dimethyl sulfoxide (DMSO; Sigma-Aldrich, Milwaukee WI)
• Mitotracker Deep Red (Invitrogen, Carlsbad CA) [mitochondrial health]
• CellEvent caspase-3/7 green detection reagent (Invitrogen, Carlsbad CA) [caspase-3/7 activation]
• 7-AAD (BD Biosciences, San Jose CA) [viability]
• V500 Syrian hamster anti-mouse CD3-e (BD Biosciences, San Jose CA) [T cells]
• APC-H7 rat anti-mouse CD19 (BD Biosciences, San Jose CA) [B cells]
• V450 rat anti-mouse CD11b (BD Biosciences, San Jose CA) [monocytes]
• PE-Cy7 rat anti-mouse Ly-6G (BD Biosciences, San Jose CA) [granulocytes]
• 4% paraformaldehyde (Thermo Fisher Scientific, Pittsburgh PA)

Procedure: Drug preparation

1. Stock solutions of doxorubicin (10 mM) and idarubicin (10 mM) are prepared in water.
2. Stock solutions of BEZ235 (25mM) and selumetinib (75 mM) are prepared in 100% dimethyl sulfoxide (DMSO).

Procedure: Splenocyte preparation and drug treatment

1. Mice are euthanized by cervical dislocation and spleens excised.
2. Splenocytes are obtained by preparing single-cell suspensions (homogenized in phosphate-buffered saline with 1% v/v fetal bovine serum using frosted glass slides) in a tissue culture dish.
3. Cells are transferred from the culture dish to a 5 mL tube and centrifuged at 400x g for 5 min, resuspended in ammonium-chloride-potassium lysing buffer to avoid red blood cell interference during flow cytometry (see below), and incubated at room temperature for 10 min in the dark.
4. Cells are centrifuged at 400x g for 5 min and resuspended in RPMI-1640 buffer supplemented with 10% FBS, 0.1% 2-mercaptoethanol, 1% v/v penicillin G/streptomycin solution.
5. Cells are then aliquoted into 96-well, round-bottom plates at a density of 100,000 cells per mL.
6. A volume of 100 µL per well of cells in supplemented media are incubated at 37°C and 5% CO₂ with either BEZ235, doxorubicin, idarubicin, or selumetinib. Each compound is plated using a nine-point logarithmic concentration scale ranging from 0.015 µM to 100 µM. (MPD has accessioned data from the following doses: 0 (control), 0.046, 0.137, 1.235, 11.1, and 100 µM. For simplicity in variable names and measurement descriptions, data from individual doses (besides control) are labeled A thru E.) Dilutions and controls are prepared to account for the inclusion of water or DMSO in the stock solution.
7. Cells are incubated for 4h.

Procedure: Splenocyte staining with fluorescent antibodies

1. At 4h post-treatment, cells are washed in PBS with 1% FBS and incubated for 30 min at 37°C and 5% CO₂ with the following indicator dyes: 125 nM Mitotracker® Deep Red for mitochondrial health, 3.75 µM CellEvent™ caspase-3/7 green detection reagent for caspase-3/7 activation, and 3.75 µL (0.19 µg) 7-AAD per 100 µL well for viability.
2. Cells are centrifuged at 400× g for 5 min, washed, and incubated at 4°C with cell indicator antibodies including 0.05 µg V500 Syrian hamster anti-mouse CD3e per 100 µL well for T-cells, 0.1 µg APC-H7 rat anti-mouse CD-19 per 100 µL well for B-cells, 0.1 µg V450 rat anti-mouse CD-11b per 100 µL well for monocytes, and 0.1 µg PE-Cy7 rat anti-mouse Ly-6G per 100 µL well for granulocytes.
3. Cells are centrifuged at 400× g for 5 minutes, washed, and fixed with 4% paraformaldehyde for 15 min at room temperature.

Procedure: Flow cytometry

1. Samples are analyzed on a FACSCanto II flow cytometer (BD Biosciences) equipped with three lasers (405 nm, 488 nm, and 640 nm).
2. The cellular populations of interest are well discriminated by forward scatter and side scatter properties. An unstained control is used to determine the threshold for samples positive for particular markers, facilitating gating as appropriate.
3. For each sample, 10,000 events are collected with the flow cytometer; data are analyzed with FlowJo version X.
4. After detection of the immune cell populations of interest, the cells positive for Mitotracker® Deep Red, activated caspase-3/7, or 7-AAD in each subpopulation are determined.

Data collected by investigator

• cell viability (percent normalized to the 0 µM dose)

*Supplemental data are available for this project (viability at all doses for B cells, T cells, and monocytes; selected IC50 values). See Wiltshire4