TITLE: CGAP Human Lung Dissociation - Tissue Stability Study
AUTHORS: Anna Wilbrey-Clark, Adam Hunter

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. Cut lung into 1cm x 1cm sections and ensure weight is 0.2-0.5g.
?. Transfer the piece of tissue to a 10cm petri dish and add ~250µl Digestion Medium to cover it.  (Digestion medium = DMEM with 1mg/ml collagenase D and 0.1mg/ml DNase I).
?. Using two scalpels, chop the piece as finely as possible.
?. Add ~2ml of Digestion Media and transfer the mashed tissue to a 15ml falcon tube using a 5ml stripette.
?. Wash the dish with 2 x 1ml of Digestion Medium, transferring it to the 15ml falcon tube with the tissue.
?. Transfer it to an incubator at 37°C for 1 hour on a rocker.  (Note softer tissue from parenchyma e.g. lower left lobe can be left for 30 minutes.  However for Tissue Stability Study the longer 1h digestion was used as airway dissociation was being tested in parallel for some donors).
?. Collect the sample and filter the cells through a 100µm nylon mesh filter into a 50ml falcon tube. Using the plunger of a syringe, repeatedly mash the filter and rinse with cold Complete Medium up to 25ml.
Complete MediaDMEM + 10% FBS
?. Spin down 360xg for 10 min at 4°C. Acceleration 4, break 2.
?. Very carefully discard the supernatant.
?. Resuspend the cell pellet in 25ml of Complete Medium.
?. Spin down 360xg for 10 min at 4°C. Acceleration 4, break 2.
?. Very carefully discard the supernatant.
?. Add 1ml of 1x Red Blood Cell Lysis solution to your cell pellet.
?. Incubate 5 min on ice with periodic agitation.
?. Add fresh cold PBS up to 10ml.
?. Spin down 360xg for 5 min at 4°C.
?. Discard supernatant.
?. Resuspend pellet in 1ml of 0.04% BSA and count cells.
?. Proceed to “MACS Live Dead Separation”.
{"blocks":[{"key":"f47p3","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[]}
?. Proceed to 10X Preparation for scRNA sequencing.
?. Ensure all unused tissue, equipment and tubes that have been in contact with primary tissue are placed into Virkon in sweetie jar for a minimum of 1 hour. After this time aspirate and disposing in relevant sharps or waste routes.
?. ABC1Material
Quantity
Supplier Info
2DMEM50mlLonza (LZBE12-614F)3FBS5mlSigma (F7524-50ML)4Collagenase (10mg/mL stock) dilute in nuclease free water
500µlSigma Aldrich (C5138-25MG)
5DNAse I (10mg/mL stock)50µlSigma Aldrich (000000011284932001)
6100mm Petri Dish
1
Corning (430591)
7Forceps
2
ThermoFisher UK Ltd (15232290)
8Scalpel
1
Swann-Morton Ltd (0507)
9100uM Cell Strainers
2
ThermoFisher UK Ltd (15380801)
1050ml Falcon Tubes
2
Falcon (352098)
112.0ml Syringe
1
ThermoFisher UK Ltd. (10673555)
1270µm Cell Strainer1
ThermoFisher UK Ltd. (15370801)
1315ml Falcon Tubes
3
Falcon (352097)
14Cold PBS
50ml
GIBCO (14190-144)
15Red Cell Lysis Buffer
2ml
Life Technologies Ltd. (00-4333-57)
160.5ml DNA LoBind Eppendorf Tubes
1
Eppendorf (0030 108.035)
17Trypan Blue
20ul
Fisher Scientific (11414815)
18C-Chips
1
Cambridge Bioscience (DHC-N01-50)
ABC1Material
Quantity
Supplier Info
2DMEM50mlLonza (LZBE12-614F)3FBS5mlSigma (F7524-50ML)4Collagenase (10mg/mL stock) dilute in nuclease free water
500µlSigma Aldrich (C5138-25MG)
5DNAse I (10mg/mL stock)50µlSigma Aldrich (000000011284932001)
6100mm Petri Dish
1
Corning (430591)
7Forceps
2
ThermoFisher UK Ltd (15232290)
8Scalpel
1
Swann-Morton Ltd (0507)
9100uM Cell Strainers
2
ThermoFisher UK Ltd (15380801)
1050ml Falcon Tubes
2
Falcon (352098)
112.0ml Syringe
1
ThermoFisher UK Ltd. (10673555)
1270µm Cell Strainer1
ThermoFisher UK Ltd. (15370801)
1315ml Falcon Tubes
3
Falcon (352097)
14Cold PBS
50ml
GIBCO (14190-144)
15Red Cell Lysis Buffer
2ml
Life Technologies Ltd. (00-4333-57)
160.5ml DNA LoBind Eppendorf Tubes
1
Eppendorf (0030 108.035)
17Trypan Blue
20ul
Fisher Scientific (11414815)
18C-Chips
1
Cambridge Bioscience (DHC-N01-50)
?. Note that this protocol was originally developed for dissociation of lung airway tissue to extract immune cells.  For the Tissue Stability Study we are using it on parenchyma tissue - the digestion time for this can be reduced to 30min.  We found that this protocol is indeed biased towards extracting immune cells.We are testing a different dissociation protocol that appears to be less immune-biased, but is still in development: dx.doi.org/10.17504/protocols.io.39ygr7w
?. Pass through a 70µm cell strainer to remove any clumps still present.