An In Vitro Method to Identify Innate and Adaptive Immune Cells Residing in Murine Lungs

Abstract

Source: Christofides, A., et al. Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung. J. Vis. Exp. (2021).

This video demonstrates an in vitro technique to identify immune cells residing in murine lungs. After isolating target immune cells from murine lung tissue, they are surface-labeled using a cocktail of fluorophore-tagged antibodies against specific markers on innate and adaptive immune cells. Additionally, monocytes, macrophages, and dendritic cells are labeled using an intracellular marker. The various immune cell types are then identified using flow cytometry.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Surgical excision and tissue preparation Euthanize the mouse by intraperitoneally injecting 1 mL of tribromoethanol (prepared according to standard protocol; Table of Materials). NOTE: CO2 asphyxiation should be avoided in lung studies as it might cause lung …

Representative Results

Figure 1: The best cellular dissociation is achieved by 5 mg/mL of collagenase 1 in prewarmed PBS + 0.5% BSA. In all different conditions, 0.2 mg of DNAse I was also included. Abbreviations: BSA = bovine serum albumin; FSC-A = peak area of forward-scattered light; L/D = live/dead staining; SF = Siglec F.

Materials

10 mL syringe plunger	EXELINT	26265
18 G needles	BD Precision Glide Needle	305165
21 G needles	BD Precision Glide Needle	305195
50 mL conical tubes	Falcon	3520
70 μm cell strainer	ThermoFisher	22363548
96-well plates	Falcon/corning	3799
ACK Lysing Buffer	ThermoFisher	A10492-01
anti-mouse CD11b	Biolegend	101215	For details see Table 2
anti-mouse CD11c	Biolegend	117339 / 117337	For details see Table 2
anti-mouse CD45	Biolegend	103115	For details see Table 2
anti-mouse CD64	Biolegend	139319	For details see Table 2
anti-mouse CD68	Biolegend	137009	For details see Table 2
anti-mouse GR-1	Biolegend	108433	For details see Table 2
anti-mouse Siglec F	Biolegend	155503	For details see Table 2
AVERTIN	Sigma-Aldrich	240486
B220	Biolegend	103228	For details see Table 2
Bovine Serum Albumin (BSA)	Sigma-Aldrich	9048-46-8
CD103	Biolegend	121405 / 121419	For details see Table 2
CD24	Biolegend	138503	For details see Table 2
CD3	Biolegend	100205	For details see Table 2
Centrifuge
Collagenase Type 1	Worthington Biochemical Corp	LS004196
CX3CR1	Biolegend	149005	For details see Table 2
DNase I	Millipore Sigma	10104159001
Ethanol
F4/80	Biolegend	123133	For details see Table 2
FcBlock (CD16/32)	Biolegend	101301	For details see Table 2
Fetal Bovine Serum	R&D Systems
Fine Serrated Forceps	Roboz Surgical Instrument Co
Foxp3 / Transcription Factor Staining Buffer Set	ThermoFisher	00-5523-00
Futura Safety Scalpel	Merit Medical Systems	SMS210
Live/Dead Fixable Far Read Dead Cell Stain Kit	ThermoFisher	L34973	For details see Table 2
MERTK	Biolegend	151505	For details see Table 2
MHC-II	Biolegend	107621	For details see Table 2
NK1.1	Biolegend	108705	For details see Table 2
Orbital Shaker	VWR	Model 200
Petri dish	Falcon	351029
Refrigerated benchtop centrifuge	SORVAL ST 16R
Small curved scissor	Roboz Surgical Instrument Co