Treatment Step Cryoprotectant Expose time Temperature
1 1 1 M Ac 1.5 min 20°C
2 2 M Ac 1.5 min 20°C
3 3 M Ac+1 M DMSO+2 M EG+2 M PG 1 min 20°C
4 3 M Ac+3 M DMSO+4 M EG+4 M PG 1 min 20°C
5 3 M Ac+5 M DMSO+5 M EG+6 M PG+15% Suc+15% H+5% PVP 1 min 0°C
6 3 M Ac+5 M DMSO+5 M EG+6 M PG+20% Suc+20% H+10% PVP 1 min 0°C
2 1 1 M Ac 1.5 min 20°C
2 2 M Ac 1.5 min 20°C
3 3 M Ac+1 M DMSO+2 M EG+1 M Gly 1 min 20°C
4 3 M Ac+3 M DMSO+4 M EG+3 M Gly 1 min 20°C
5 3 M Ac+5 M DMSO +5 M EG+5 M Gly +15% Suc+15% H+5% PVP 1 min 0°C
6 3 M Ac+5 M DMSO+5 M EG+5 M Gly+20% Suc+20% H+10% PVP 1 min 0°C
3 1 1 M Ac 1.5 min 20°C
2 2 M Ac 1.5 min 20°C
3 3 M Ac+1 M DMSO+2 M EG+2 M MeOH 1 min 20°C
4 3 M Ac+3 M DMSO+4 M EG+4 M MeOH 1 min 20°C
5 3 M Ac+5 M DMSO +5 M EG+6 M MeOH+15% Suc +15% H + 5% PVP 1 min 0°C
6 3 M Ac+5 M DMSO+5 M EG+6 M MeOH +20% Suc+20% H+10% PVP 1 min 0°C
4 1 1 M Ac 1.5 min 20°C
2 2 M Ac 1.5 min 20°C
3 3 M Ac+2 M EG+2 M PG+1 M Gly 1 min 20°C
4 3 M Ac+4 M EG+4 M PG+3 M Gly 1 min 20°C
5 3 M Ac+5 M EG+6 M PG+5 M Gly+15% Suc+15% H+5% PVP 1 min 0°C
6 3 M Ac+5 M EG+6 M PG+5 M Gly+20% Suc+20% H+10% PVP 1 min 0°C
5 1 1 M Ac 1.5 min 20°C
2 2 M Ac 1.5 min 20°C
3 3 M Ac+2 M EG+2 M PG+2 M MeOH 1 min 20°C
4
5
6
3 M Ac +4 M EG+4 M PG+4 M MeOH 1 min 20°C
3 M Ac+5 M EG+6 M PG+6 M MeOH+15% Suc+15% H+5% PVP 1 min 0°C
3 M Ac+5 M EG+6 M PG+6 M MeOH+20% Suc+20% H+10% PVP 1 min 0°C
6 1 1 M Ac 1.5 min 20°C
2 2 M Ac 1.5 min 20°C
3 3 M Ac+2 M PG+1 M Gly+2 M MeOH 1 min 20°C
4 3 M Ac+4 M PG+3 M Gly+4 M MeOH 1 min 20°C
5 3 M Ac+6 M PG+5 M Gly+6 M MeOH+15% Suc+15% H+5% PVP 1 min 0°C
6 3 M Ac+6 M PG+5M Gly+6 M MeOH+20% Suc+20% H+10% PVP 1 min 0°C
Table 1: Steps of the cryoprotectant incorporation protocol.
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