Waarom is het testen van de nulhypothese belangrijk voor PD-L1 flowcytometrie?

Het testen van de nulhypothese bij PD-L1 flowcytometrie maakt een objectieve evaluatie mogelijk van of de waargenomen marker-expressie significant verschilt tussen experimentele groepen, wat bijdraagt aan een strikte targetvalidatie en het aantal fout-positieven in de vroege ontdekkingsfase vermindert.

Hoe past de isolatie van de onafhankelijke variabele in de workflows voor longcel-suspensies?

Het isoleren van variabelen, zoals enzymconcentratie en incubatietijd, garandeert dat veranderingen in de celopbrengst of markerexpressie kunnen worden toegeschreven aan de experimentele omstandigheden, wat de reproduceerbaarheid en interpreteerbaarheid in discovery-pipelines verbetert.

Wat maken kwantitatieve flowcytometrie-metingen mogelijk in longmodellen?

Kwantitatieve flowcytometrie biedt nauwkeurige gegevens over celpopulaties en markerexpressie, wat vergelijkende analyses tussen verschillende condities mogelijk maakt en datagestuurde beslissingen in immuno-oncologisch onderzoek ondersteunt.

Waarom zijn replicatievereisten essentieel voor protocollen voor de preparatie van longcellen?

Replicatie zorgt ervoor dat de resultaten van celisolatie en flowcytometrie consistent zijn over experimenten en teams heen, wat cross-functionele samenwerking en een betrouwbare voortgang van preklinische programma's faciliteert.

Welke statistische analysemogelijkheden zijn vereist vóór de implementatie van flowcytometrische PD-L1-studies?

Een robuuste statistische analyse is nodig om flowcytometriegegevens te interpreteren, de significantie van verschillen in markerexpressie te beoordelen en go/no-go-beslissingen in targetvalidatie-workflows te sturen.

Mouse Lung Preparation for Flow Cytometry: Generating Cell Suspension from Lung Tissue for Flow Cytometric Analysis

JoVE Encyclopedia of Experiments

Kankeronderzoek

0 weergaven • 3:48 min • April 30th, 2023

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- Begin by taking a euthanized mouse and secure it on a dissection board with lab tape. Use scissors to cut open the skin. Make a vertical incision through the diaphragm and cut ribs to reach the thoracic cavity exposing the heart and lungs. Next, make a small incision in the left ventricle of the heart. Subsequently, locate the right ventricle and insert a syringe containing PBS into the ventricle's lumen. Perfuse PBS through the right ventricle letting the blood flow out of the premade incision at the left ventricle till the lungs turn white. Take out the lung tissue containing tumor nodules and mince into small pieces.

Incubate the pieces in the lung digestion buffer. Collagenase in the lung digestion buffer helps isolate cells, and DNAse helps eliminate DNA from the dead cells. Strain the resulting suspension through a cell strainer to remove clumps and make the cell suspension uniform. Centrifuge and resuspend the pellet in the ammonium chloride potassium buffer to lyse residual erythrocytes. Centrifuge to collect the WBCs and tumor cells in the pellet. Resuspend the pellet in PBS supplemented with FCS, to maintain cell stability. In the following protocol, we will demonstrate the preparation of lung cell suspension for flow cytometric analysis.

After the appropriate experimental endpoint, soak the carcass in 70% ethanol and secure the animal to a dissection board. Make a ventral midline incision and gently invert the skin to expose the thoracic wall muscles and the abdominal organs. Use scissors to puncture the diaphragm and to cut the ribs exposing the thoracic cavity. Cut a small opening in the left ventricle and use a 27 gauge needle to perfuse the lung three times through the right ventricle with 6 to 8 milliliters of ice cold PBS per infusion. After the last perfusion the lungs should be completely clear of blood and appear white. After harvesting, use scissors to mince the lung tissue into small pieces.

Transfer the lung fragments into a 2 milliliter microcentrifuge tube containing 1.5 milliliters of lung digestion buffer for a 30 to 60 minute incubation at 37 degrees Celsius with shaking. At the end of the digestion, transfer the cell suspension through a 70 micron cell strainer into a 50 milliliter tube, and use the end of a sterile 10 milliliter syringe plunger to press any remaining tissue fragments through the filter. Rinse the strainer with 15 milliliters of PBS supplemented with 2% FCS, and collect the cells by centrifugation. Resuspend the pellet in 1 milliliter of ammonium chloride potassium lysis buffer for a 5 minute incubation at room temperature, and centrifuge the cells again. Then resuspend the white blood cell pellet in 1 milliliter of PBS supplemented with 2% FCS, and stain the cells for flow cytometric analysis according to the experimental protocol.