Table 6.
Variability in murine bone marrow stromal cell isolation protocols.
| Samples | Isolation medium | RBC lysis | Enzymatic digestion | Depletion/enrichment | Application | References |
|---|---|---|---|---|---|---|
| Flushed BM | DMEM/F12, 20% FBS, P/S, 2 mM L-glutamine, 0.1 mM NEAA, 3 mM sodium pyruvate | – | – | – | Cell culture | (147) |
| PBS, 2%FBS, 2 mM EDTA | – | 3 mg/ml col. I + 4 mg/ml Dispase (15 min 37°C) |
– | Cell culture | (148) | |
| Leibovitz's L-15 medium, 1 mg/ml BSA, 10 mM HEPES, 1% P/S | 0.8% NH4Cl | 0.005% trypsin + 0.002% EDTA + 0.25 mg/ml col. IV (4 min 37°C) | Anti-CD45, Nestin-GFP+ FACS | Flow cytometry | (149) | |
| HBSS, 2%FBS | – | DNase I + col. IV or + liberaseDL (15 or 20 min 37°C) | – | Flow cytometry | (27, 150) | |
| DMEM, 15% FBS, 2 mM L-Glutamine, 1% P/S, 3.7 g/l NaHCO3 | – | – | – | Cell culture | (151) | |
| α-MEM, 10% FBS, 1% P/S | – | – | – | Cell culture | (152) | |
| α-MEM, 15% FBS, 1% P/S, 2.2/l NaHCO3 | – | – | – | Cell culture | (153) | |
| α-MEM | – | – | – | Cell culture | (154, 155) | |
| RPMI-1640, 10% FBS, 1% P/S | – | – | – | Cell culture | (156) | |
| - | - | Trypsin (2 min 37°C) | – | Cell culture | (84) | |
| Long term medium | – | – | – | Cell culture | (157) | |
| RPMI-1640, 20% FBS, 2 mM glutamine, 1% P/S | – | – | – | Cell culture | (158) | |
| Centrifuged BM | N/A | RBC lysis buffer | – | – | BMAd isolation | (56) |
| N/A | – | – | – | Cell culture | (135, 159) | |
| Crushed long bones | PBS | – | col. (20 min 37°C) | – | Flow cytometry | (28) |
| Flushed and cut long bones | α-MEM, 10% FBS | – | 1 mg/ml col. II (1–2 h 37°C) | – | Cell culture | (160) |
| Cut and washed long bones | DMEM | H2O 6 s | 0.2% col. (1 h 37°C) | – | Flow cytometry | (141) |
| N/A | – | 5% col. | Anti-CD45, anti-Ter119, anti-CD31 | Cell Culture | (161) | |
| Cut long bones | PBS, 20% FBS | ACK | 0.5% col. II (1 h 37°C) | – | Flow cytometry | (5) |
BSA, bovine serum albumin; Col., collagenase; DMEM, dulbeco's modified eagle medium; FBS, fetal bovine serum; RBC, red blood cell; RPMI, L-glutamine, phenol red, reduced serum; P/S, penicillin/streptomycin; NEAA, non-essential amino acids.