Extraction conditions withstood by strings in live cells at 4°C
| No. | Extraction | Rationale of use |
| 1 | 100 mM NaOAc, pH 5.5, 5 min | Disrupt pH-dependent interactions |
| 2 | 100 mM Na2CO3, pH 11, 3 min | Disrupt pH-dependent interactions |
| 3 | 10 µg/ml PPS, 30 min, or 500 µg/ml PPS, 1 h, or 1 µg/ml HM, 90 min (in PBS) | Compete with interactions with sulfated glycosaminoglycans |
| 4 | 10 µg/ml PPS in 100 mM Na2CO3, 3 min | Combines conditions 2 and 3 |
| 5 | 2 M NaCl, 2 min | Disrupts electrostatic interactions including interactions with GAGs |
| 6 | 0.1% Triton X-100 in PBS, 5 min | Does not extract GPI-APs (Mayor and Maxfield, 1995) |
| No. | Extraction | Rationale of use |
| 1 | 100 mM NaOAc, pH 5.5, 5 min | Disrupt pH-dependent interactions |
| 2 | 100 mM Na2CO3, pH 11, 3 min | Disrupt pH-dependent interactions |
| 3 | 10 µg/ml PPS, 30 min, or 500 µg/ml PPS, 1 h, or 1 µg/ml HM, 90 min (in PBS) | Compete with interactions with sulfated glycosaminoglycans |
| 4 | 10 µg/ml PPS in 100 mM Na2CO3, 3 min | Combines conditions 2 and 3 |
| 5 | 2 M NaCl, 2 min | Disrupts electrostatic interactions including interactions with GAGs |
| 6 | 0.1% Triton X-100 in PBS, 5 min | Does not extract GPI-APs (Mayor and Maxfield, 1995) |
(Nos. 1–5) Live ScGT1 cells on Permanox slides were chilled to ice temperature, treated as detailed, and then fixed and stained with 8B4, except for HM-treated cells, which were immunostained on ice before fixation. (No. 6) For the Triton X-100 treatment, cells chilled to 4°C were first immunostained, then incubated with Triton X-100, and then fixed.