an in vitro method to identify innate and adaptive immune cells residing in murine lungs

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

Transfer the lung to a Petri dish. Mince it using two fine scalpels, and then place it into a 50-milliliter conical tube. Add 5 milliliters of digestion buffer to wash the plate. Secure the lid of the tube, and digest the lung for 30 minutes on an orbital shaker at a speed of 150 rotations per minute at 37 degrees Celsius. Stop the reaction by adding 10 milliliters of cold BSA buffer.
After digestion, mix and dissolve the lung pieces using an 18-gauge needle. Place a 70-micrometer filter strainer on top of a new 50-milliliter conical tube, and transfer the digested lung mixture into the strainer.
Use the rubber side of a 10-milliliter syringe plunger to smash the remaining lung pieces on the filter, and wash it with BSA buffer. Centrifuge the single-cell suspension at 350 g for 8 minutes at 7 degrees Celsius. Discard the supernatant, and resuspend the cells in 1 milliliter of ACK lysis buffer. Mix the suspension using a 1-milliliter pipette, and incubate it for 90 seconds at room temperature.
Add 10 milliliters of cold BSA buffer to the reaction mixture, and centrifuge it at 350 g for 7 minutes at 4 degrees Celsius. Discard the supernatant. Resuspend the pellet in staining buffer, and count the cells using a hemocytometer. Resuspend the cells at a concentration of 5 million cells per milliliter for surface staining.
Transfer 1 million cells in 200 microliters per well into a 96-well plate, and centrifuge the plate at 350 g for 7 minutes at 4 degrees Celsius. Prepare a flow cytometry block solution by diluting anti-16/32 antibody in staining buffer.
Resuspend the cells in 50 microliters of the flow cytometry block solution. Incubate the suspension for 15 to 20 minutes at 4 degrees Celsius, or on ice. Then, add 150 microliters of staining buffer to the plate, and centrifuge it at 350 g for 5 minutes at 4 degrees Celsius.
Prepare surface antibody solution by diluting the surface antibodies in staining buffer. Resuspend the cells in 50 microliters of the surface antibody solution, and incubate the plate at 4 degrees Celsius in the dark. Then, wash the cells twice with staining buffer.
Prepare the fixation and permeabilization buffer by mixing three parts fixation and permeabilization diluent, and one part staining buffer. Resuspend the cells in 50 microliters of the preprepared buffer per well of the 96-well plate, and incubate them for 20 to 25 minutes at 4 degrees Celsius in the dark.
Dilute the permeabilization buffer with purified deionized water to prepare one times permeabilization buffer, and use it to wash the cells. Prepare an intracellular antibody solution by diluting with 1 milliliter of permeabilization buffer. Resuspend the cells in 50 microliters of the diluted intracellular antibody solution per well of the 96-well plate, and incubate for 40 minutes at 4 degrees Celsius in the dark.
Wash the cells with permeabilization buffer, then with staining buffer. After the final wash, resuspend the cells in 200 microliters of staining buffer. Acquire a minimum of 1.5 million cells per sample on the flow cytometer.

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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
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	<li>Euthanize the mouse by intraperitoneally injecting 1 mL of tribromoethanol (prepared according to standard protocol;&#160;Table of Materials). 
	NOTE: CO2&#160;asphyxiation should be avoided in lung studies as it might cause lung ...

Source: Christofides, A., et al. <a href="https://app.jove.com/v/62985">Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung.</a> 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.

<img alt="Figure 1" src="/files/ftp_upload/22141/22141_Figure1.jpg" /> 
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.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Take minced murine lung tissue in a digestion buffer and incubate at a physiological temperature.
Buffer enzymes break down the tissue matrix, loosening the cells.
Triturate to dissociate the cellular mass, and filter the suspension using a strainer to isolate the single cells.
Centrifuge to remove the supernatant with tissue debris. Incubate the cells with a lysis buffer to lyse the red blood cells.
Centrifuge to discard the cellular debris-containing supernatant. Resuspend the cells and transfer them to a microplate.
Add antibodies to block the Fc receptors, preventing background staining.
Introduce a fluorophore-labeled antibody cocktail targeting specific surface markers on the innate immune cells &#8212; monocytes, macrophages, dendritic cells, natural killer cells, neutrophils, eosinophils, and adaptive immune cells &#8212; B and T lymphocytes.
Fix and permeabilize the cells. Add fluorophore-labeled antibodies specific for an intracellular marker on monocytes, macrophages, dendritic cells, and eosinophils.
Using flow cytometry, classify the labeled immune cells.

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

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