Nanovial Multicell Experiment Record
CF030 / CF031 · Partillion Bioscience
Experiment Info
Experiment ID
Date
Operator
Protocol Reference
Instrument
Module 1
Nanovial Modification
Steps 1.1 – 1.5 · ~1.5 hrs · Recommended 1–2 days before assay
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⚙
Nanovial Parameters
Lot Number
Size
50 µm EZM™
35 µm EZM™
Total NVs to Modify
(count)
NV Stock Conc.
(NVs/mL)
NV Volume to Use
(µL, calc)
1.1
Wash Nanovials
Pre-coat a 5 mL tube with 1X Wash Buffer
Pipette up/down 10× to resuspend NVs; transfer to pre-coated tube
Add 2 mL 1X Wash Buffer; centrifuge 300×g, 3 min; remove supernatant; resuspend in 1X Wash Buffer to original volume
1.2
Streptavidin Coating
📐 Target: 200 µg/mL final. Prepare 2X solution at 400 µg/mL in equal volume to NVs.
SA Species / Source
SA Stock Conc.
(µg/mL)
SA Vol. Needed
(µL, calc)
SA Vol. Override
(µL)
Wash Buffer Vol.
(µL, calc)
Prepare 2X SA solution (400 µg/mL) in equal volume to NVs
Add equal volume of 2X SA to washed NVs; mix 10× by pipetting
Incubate 30 min, RT, rotator <60 rpm
1.3
Post-Streptavidin Wash
Top up tube with 1X Wash Buffer to 5 mL mark
Centrifuge 300×g, 3 min
Wash SA-coated NVs twice with 1 mL 1X Wash Buffer
Resuspend to original NV volume in 1X Wash Buffer (pipette 10×)
1.4
Prepare Capture Antibodies
📐 Target: 20 µg/mL final. Prepare 2X mix at 40 µg/mL in equal volume to NVs. Formula: Vol (µL) = (40 µg/mL × NV Vol µL) / Stock conc.
Antibody Name
Clone / Catalog #
Stock Conc. (µg/mL)
Vol. Needed (µL, calc)
Override (µL)
+ Add Antibody
Spin down biotinylated antibodies 14,000×g, 10 min; pipette from top
Prepare 2X antibody mix (40 µg/mL each) in equal volume to NVs
Add equal volume 2X antibody mix to SA-NVs; mix 10× by pipetting
Incubate ≥30 min, RT, rotator <60 rpm
1.5
Post-Antibody Modification Wash
Top up with 1X Wash Buffer to 5 mL; centrifuge 300×g, 3 min
Wash modified NVs twice with 1 mL 1X Wash Buffer
Wash once more with 1 mL Cell Culture Media; centrifuge 300×g, 3 min
Resuspend to original NV volume in fresh Cell Culture Media (pipette 10×)
Aliquot 150 µL modified NVs per ratio sample into pre-coated 1.5 mL tubes
Aliquot 25 µL for Unstained Modified NV control; 25 µL for Stained Modified NV control → keep on ice
Module 2
Assay
Steps 2.1 – 3.3 · Cell Loading, Incubation, Wash
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Cell Identities & Working Concentrations
Cell A (Captured)
Cell A Identity
Working Conc.
(cells/mL)
Staining Method
Pre-load viability dye (e.g. Calcein AM)
Post-load antibody staining
Cell surface marker only
None / unlabeled
Dye / Marker
Cell B (Reporter)
Cell B Identity
Working Conc.
(cells/mL)
Staining Method
Post-load antibody staining
Pre-load viability dye
Cell surface marker only
None / unlabeled
Dye / Marker
Cell Culture Media
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Conditions Table
Enter # NVs per condition (defaults to Module 1 total), then Cell A:NV and Cell B:NV ratios. Volumes are calculated from working concentrations set above.
Condition Name
# NVs
NV Vol. (µL)
Cell A : NV
Cell A Vol. (µL)
Cell B : NV
Cell B Vol. (µL)
Notes
Stained Modified NVs (ctrl)
— no cells —
Unstained Modified NVs (ctrl)
— no cells —
+ Add Condition
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)
📝
Overall Experiment Notes
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Experiment ID:
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