Overview of experimental protocols applied to generate viable pulmonary single-cell suspensions; cell isolation procedures corresponding to the interspecies datasets to dissociate lung tissue into a single-cell suspension. Enzymatic digestion and mechanical processing steps applied to dissociate lung tissue into a single-cell suspension vary between species.
| Human lung tissue (Charité) | Murine lung tissue (Charité) | Hamster lung tissue (Nouailles et al. [18]) | Human lung tissue (Travaglini et al. [24]) | Monkey lung tissue (Speranza et al. [21]) | Rat lung tissue (Raredon et al. [25]) | Pig lung tissue (Raredon et al. [25]) | |
| Treatment | Mock infection | Mock infection | Heparinisation | ||||
| 1st enzymatic digestion | Intra-alveolar vasculature | Intra-alveolar vasculature | Intra-alveolar | ||||
| Enzymes | Dispase (50 U·mL−1) | Collagenase/dispase (1 mg·mL−1) elastase (3 U·mL−1) DNase (20 U·mL−1) | Collagenase/dispase (1 mg·mL−1) elastase (3 U·mL−1) DNase (20 U·mL−1) | ||||
| Temperature | 22°C | 37°C | 37°C | ||||
| Duration | 5 min | ||||||
| 1st mechanical dissociation | Mincing with scissors | Clapping with tweezers | Clapping with tweezers | Dissection and mincing | Dissociation in rocker | Dissociation in rocker | |
| 2nd enzymatic digestion | Distal alveolar tissue | Full lung tissue | Lobus caudalis | Distal alveolar tissue | Lung sections | Full lung tissue | Distal lung regions |
| Enzymes | Collagenase I (500 U·mL−1) DNase I (1 U·mL−1) dispase (5 U·mL−1) | Collagenase CLS II (750 U·mL−1) DNase I (1 mg·mL−1) | Collagenase CLS II (750 U·mL−1) DNase I (1 mg·mL−1) Dispase (50 U·mL−1) | Liberase DL (400 µg·mL−1) elastase (100 µg·mL−1) | Liberase (0.1 mg·mL−1 DNase I (0.02 mg·mL−1) | Collagenase/dispase (1 mg·mL−1) elastase (3 U·mL−1) DNase (20 U·mL−1) | Collagenase/dispase (1 mg·mL−1) elastase (3 U·mL−1) DNase (20 U·mL−1) |
| Temperature | 37°C | 37°C | 37°C | 37°C | 37°C | 37°C | 37°C |
| Duration | 60 min | 30 min | 30 min | 30 min | NR | 20 min | 40 min |
| 2nd mechanical dissociation | Shear force via syringe | Shear force via pipette | GentleMACS dissociator | Pressed through sieve# | Pressed through sieve# | ||
| 2nd incubation | 30 min nutator at 37°C | ||||||
| 3rd dissociation | GentleMACS dissociator+DNAse I (100 µg·mL−1) | ||||||
| 1st cell filtration | 70 µm cell strainer | 70 µm cell strainer | 70 µm cell strainer | 100 µm cell strainer | 100 µm cell strainer | 70 µm cell strainer | 70 µm cell strainer |
| Red blood cell lysis | Yes | Yes | Yes | Yes | Yes | Yes | Yes |
| 2nd cell filtration | 40 µm cell strainer | 70 µm cell strainer | 40 µm cell strainer | 70 µm cell strainer | 40 µm cell strainer | 40 µm cell strainer | |
| Isolation buffer | RPMI, 10% FCS, 1% glutamine | 1× PBS, 0.5% BSA | 1× PBS, 0.5% BSA | 1× PBS, 5% FBS | 1× PBS, 0.1% BSA | 1× PBS, 0.01% BSA | 1× PBS, 0.01% BSA |
| Centrifugation force | 360×g (10 min, 4°C) | 300×g (6 min, 4°C) | 350×g (6 min, 4°C) | 300×g (5 min, 4°C) | NR | 300×g (3 and 5 min) | 300×g (3 and 5 min) |
| Single-cell processing | 10× Genomics | 10× Genomics | 10× Genomics | 10× Genomics/SMART-seq2 | 10× Genomics | 10× Genomics | 10× Genomics |
DL: dispase low; FCS: fetal calf serum; BSA: bovine serum albumin; FBS: fetal bovine serum; NR: not reported. #: the sieve was additionally rinsed with DMEM containing 10% FCS, 1% penicillin/streptomycin, 1% amphotericin and 0.1% gentamicin.