Cell dissociation (Yale)

Rat Lung scRNAseq Dissociation

Last updated 2026-03-30 by MSBR

Raredon Lab

Animals: SD Female and Male ~220/250grams

Anesthetic: KX 75mg/5mg per kg body weight

Pre-Op: Intraperitoneal heparin 0.15ml at 1000U/ml (critical to allow blood clearance from lung tissue)

Setting: Biosafety hood for sterile cell isolation. Benchtop OK for non-sterile.

Surgery:

Abdominal incision

Diaphragm incision and tension

Chest clamshell

Thymic dissection to expose great vessels

Neck dissection to expose trachea

Cannulate trachea with barbed Y-fitting 1/16” ID, test with 10ml syringe, inflate/deflate lungs a few times GENTLY – oxygenation will buy a few more minutes of heartbeat

Cannulate pulmonary artery under stopcock-restricted gravity-driven dribble:

Hep 100U/ml + (Sodium Nitroprusside 1 mg/ml diluted 100:1) 

Avoid air embolus during cannulation

Cut off apex of heart to allow free left ventricle outflow

Open stopcock to full gravity flow

Gently ventilate the lungs a few times during perfusion to break up microclots

Lungs should go uniform paper-white

Tracheal barrier should be maintained (no froth in airway syringe)

The goal in the above steps is to minimize the circulating blood fraction in the resulting dissociation. A well-executed perfusion under heparin can reduce the fraction to <10% of total cells.

Tissue Dissociation:

Dissect heart/lung bloc to large dry sterile petri dish

Switch gravity-driven perfusion solution to the below enzyme mixture

25 ml enzyme solution @ 37C:

            25 ml DMEM HG

            1mg/ml Collagenase/Dispase (Roche)

            3 U/ml Elastase (Worthington)

            20 U/ml DNAse (Worthington)

Avoid/catch and correct any air bubbles in the lines

Collect left-ventricular effluent from petri dish into a 10 ml syringe

Infuse enzyme effluent x3 times into the trachea

If the above fails, you may inject the tissue with enzyme using a 25 gauge syringe

The goal is to completely saturate the tissue with warm enzyme and allow to incubate at 37C BEFORE mechanical dissociation – this preserves ATI and alveolar yield.

Dissect and discard heart tissue and large airways

Place intact, enzyme-saturate lobes on a rocker at 37C for 20 minutes in 50 ml conical.

Pig and human tissues may require up to 40 minutes.

Following this incubation, the tissue should be visibly compliant/gelatinous/friable

Squish the tissue through a sterile stainless steel kitchen strainer using a sterile metal spatula, above a large petri dish

Parenchymal tissue will turn to a cloud below the strainer

Larger collagenous structures will remain within the strainer. More aggressive grinding = more conducting structure cells.

Single-Cell Suspension:

Rinse strainer with 20 ml ice-cold media containing FBS or similar enzyme-quenching reagent:

            DMEM HG

            10% FBS

Optional, if planning downstream cell culture:

            1% P/S

            1% Amp

            0.1% Gent

Collect all material outside of strainer, dissociated cells and solubilized matrix. Then, one ice / at 4C:

[being cold here is critical for cell viability]

1) Centrifuge 5 min @ 300g, remove supernatant [this removes the majority of the solubilized matrix]

Add red cell lysis buffer 1:1 with pellet volume, 120 seconds at RT, dilute with ~25 ml of 0.04% BSA in PBS (0.1mg/ml). [this lyses red blood cells so that they don’t distort the downstream cell counts]

2) Centrifuge 5 min @ 300g, remove supernatant

Resuspend in 0.04% BSA. Filter through 70um filter, rinse filter. [this removes large cell clumps]

3) Centrifuge 3 min @ 300g, remove supernatant. 

Resuspend in 20ml 0.04% BSA in PBS, pass through 40um filter, rinse filter with 5 ml, pass through 40um filter, rinse filter with 5 ml [this removes most doublets and brings to a truly single-cell suspension]

Count cells – ALL CELLS MUST BE COUNTED FOR SINGLE-CELL DILUTIONS, NOT JUST LIVE CELLS

[if live-cell-only counts are used for single-cell dilution calculations, the suspension will have an unacceptable doublet rate and will not yield usable data]

Record cell viability and observed doublet rate. Target is >80% viable with <5% doublets.

Serial dilute to final concentration of 100,000 cells/ml for single-cell sequencing in 0.01% BSA in PBS. 

Place in conical on ice for transfer to library-prep / cytospin / culture / freezing / bulk RNA isolation.