Single-Cell Western Blotting to Determine Cell Heterogeneity

Abstract

Source: Wang, L., et al., Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages. J. Vis. Exp. (2019).

The video describes a single-cell western blotting technique that identifies the composition of individual cells captured on a single-cell western blotting chip in a heterogeneous suspension. The technique detects the fluorescence intensity of the fluorophore tagged to a protein marker specific to the target cell.

Protocol

1. Determining Cell Composition in the Cell Suspension Using Single-cell Western Blot (scWB) Analysis Rehydrate the scWB chip before use in 15 mL of 1x suspension buffer in a Petri dish for at least 10 min at RT. Add 1 mL of the diluted single-cell suspension (10,000-100,000 cells/mL) to the rehydrated chip. Set the surface onto the bottom of a 10 cm Petri dish. Ensure that the surface is set flat. Cover the Petri dish with a lid to prevent drying and let the cells settle for 5-15 min by a…

Materials

10 cm Petri dish	griner bio-one	664 160
10x suspension buffer	proteinsimple	R101
15 cm Petri dish	Falcon	353025
2-Mercaptoethanol	MP BIOMEDICALS	2194705
5% washing buffer	proteinsimple	R252
Antibody diluent	proteinsimple	042-203
Bench Top Centrifuge	Beckman Coulter		Microfuge 22R Centrifuge
Bovine Serum Albumin	Sigma-Aldrich	A7906-100G
Calcein AM	ThermoFisher	C3099	1 mL
CD11b	Novus Biologicals	NB110-89474
CD45 (D4H7K) rabbit mAb	Cell Signal Technologies	727987S
CD68/SR-D1 (FA-11)	Novus Biologicals	NBP2-33337SS
Cellometer Vision	Nexcelom
Dispase	BD	354235	100 mL
Dissecting scissor
Donkey anti-Rabbit IgG secondary antibody [NL557]	Novus Biologicals	NL004	NL 557 conjugate
Donkey anti-Rat IgG (H+L) secondary antibody [DyLight 650]	Novus Biologicals	NBP1-75655	DyLight 650 conjugate
F4/80 (CI-A3-1)	Novus Biologicals	NB600-404SS
Heat Block	effendorf	22331	Thermomixer
Lysis/Running buffer	proteinsimple	R200
Matrigel Matrix (Growth Factor Reduced)	BD	354230	10 ml
Microarray scanner	PerkinElmer	ScanArray Gx
Microcentrifuge tube	Fisher Scientific	05-408-129
Microscope	ThermoFisher	Evos fl
Milo	proteinsimple		Single cell western
Needle	BD	305111	26 G
Parafilm
Probing chamber	proteinsimple	035-020
Recombinant mouse CCL2/JE/ MCP-1 protein	R&D	479-JE-050
scWEST chip	proteinsimple	C300
Shaker	Bioexpress		Gene mate orbital shaker
Single cell western chip canister	proteinsimple	035-118
Slide spinner	Labnet	C1303-T
Sodium Dodecyl Sulfate (SDS)	Fisher Scientific	BP 166-500
Syringe	BD	309602	1 mL
Tris-Hydrochloride	Fisher Scientific	BP 153-500
Tweezer	proteinsimple	035-020
Water bath	ThermoFisher