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Cell Identities & Working Concentrations

Cell A (Captured)

Cell A Identity

Working Conc. (cells/mL)

Staining Method

Dye / Marker

Cell B (Reporter)

Cell B Identity

Working Conc. (cells/mL)

Staining Method

Dye / Marker

Cell Culture Media

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Conditions Table

Cell A:NV and Cell B:NV ratios are auto-calculated from counts and NV number. Ratios update as you type.

Condition Name	# NVs	Cell A Count	Cell A:NV Ratio	Cell A Vol. (µL)	Cell B Count	Cell B:NV Ratio	Cell B Vol. (µL)	Notes
Stained Modified NVs (ctrl)		—			—
Unstained Modified NVs (ctrl)		—			—

2.1

Preparation of Cell A

Count cells; confirm viability >90%
Obtain ~2M viable cells + 25–50% overage for wash losses
Wash 2× with 1X Staining Buffer (1 mL per 1M cells); centrifuge 300×g, 4 min
Resuspend to 1M cells/mL in stain (or Cell Culture Media if surface markers available)

2.2

Staining of Cell A (if pre-load dye)

Prepare Calcein AM per manufacturer (titrate if needed); target 25 nM final
Resuspend cells in diluted dye (1 mL per 1M cells); incubate 30 min, 4°C, dark
Wash with 1X Staining Buffer; centrifuge 300×g, 4 min; count and adjust to 1M viable cells/mL
Final resuspension in Cell Culture Media at 1M cells/mL → proceed to loading immediately

2.3

Loading of Cell A

Pipette appropriate Cell A volumes per condition (see table); mix before each aliquot
After adding cells, pipette up/down 10× to mix cells and NVs
Centrifuge 100×g, 1 min; incubate on ice, 15 min
Resuspend; centrifuge 100×g, 1 min; incubate on ice, 15 min (second round)
Reserve remaining stained Cell A for single-color control

2.4

Preparation of Cell B

Count cells; obtain required quantity + 25–50% overage
Wash with 1X Staining Buffer (1 mL per 1M cells); centrifuge 300×g, 10 min
Resuspend to 4M viable cells/mL in 1X Staining Buffer; count and adjust
Final wash; resuspend to 4M viable cells/mL in Cell Culture Media → load immediately

2.5

Loading of Cell B

Pipette appropriate Cell B volumes per condition; mix before each aliquot
Pipette up/down 10× to mix cells and NVs
Centrifuge 100×g, 1 min; incubate on ice, 15 min
Resuspend; centrifuge 100×g, 1 min; incubate on ice, 15 min (second round)

3.1

Wash Cell-Loaded Nanovials

Allow Cell Culture Media to warm to RT
Centrifuge all tubes 300×g, 3 min; discard supernatant (do NOT aspirate pellet)
Add 1 mL Cell Culture Media per sample; pipette up/down 10×

3.2

Cell Incubation

Incubation Time (min)

Temperature (°C)

CO₂ (%)

Transfer tubes to CO₂ incubator at 37°C for incubation period
During incubation: prepare staining cocktail (steps 3.4 and 3.5)

3.3

Post-Incubation Wash

Wash samples twice with 1 mL 1X Staining Buffer; 300×g, 3 min, RT
Resuspend NV pellet in staining cocktail (ratio samples) or staining solution (controls)

3.4

Staining of Cell B and DAPI

Staining cocktail: 50 µL RB705 + 2 µL DAPI + 948 µL 1X Staining Buffer (per 1000 µL). Add 100 µL per ratio sample.

Cell B Marker

DAPI Stock Conc. (mg/mL)

Staining cocktail notes

Prepare DAPI to 20 mg/mL stock
Prepare staining cocktail in separate tube
Add 100 µL cocktail per ratio sample; pipette 10×; incubate 30 min on ice, dark

3.5

Modified Nanovial Control Staining

Prepare diluted RB705: 5 µL stock + 95 µL Staining Buffer (10 µg/mL final)
Add 100 µL to Stained Modified NV control; pipette 10×; 30 min on ice, dark
Unstained Modified NV control: resuspend in 1X Staining Buffer only (no stain)

3.6

Sample Preparation for Flow Cytometry

After 30 min incubation, wash all samples 2× with 1 mL 1X Wash Buffer (300×g, 3 min)
Resuspend all samples in 800 µL 1X Wash Buffer; transfer to pre-coated flow tubes (NOT filtered)

4.1

Flow Cytometry — Gating Strategy

Instrument

Nozzle Size (µm)

Software

Vortex each sample immediately before acquisition
Gate A: SSC-A vs LightLoss-A → Nanovial events
Gate B (from Gate A): SSC-A vs DAPI-A → DAPI-negative (live) events
From Gate B: RB705-A vs Calcein-AM → Q1 (Cell B+), Q2 (A+B+), Q3 (A+), Q4 (Empty). Target ≥5000 Q2 events
Export quadrant counts for each sample
Perform compensation if necessary; adjust gains

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Overall Experiment Notes

Nanovial Multicell Experiment Record

Experiment Info

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