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我正在用的，仅供参考
Flow cytometry analysis of exosomes-bound beads
1. Incubate 30 µg of exosomes or 30 µg BSA (negative control) in a 10 µl volume of beads at RT for 15 min with continuous rotation.
2. This suspension was diluted to 1ml with PBS and incubate the mixture at RT for a further 2 h or at 4 ℃ overnight with agitation.
3. Terminate the coupling reaction by the addition of 100 mM glycine and 2% BSA in PBS and left rotating for 30 min at RT.
4. Exosomes-bound beads were washed once in 2% BSA in PBS and centrifuged for 1 min at 14,800g, blocked with 10% BSA with rotation at room temperature for 30 min, washed a second time in 2 % BSA and centrifuged for 1 min at 14,800g, and incubated with anti-X during 30 min rotating at 4 ℃. .
5. Beads were centrifuged for 1 min at 14,800g, the supernatant was discarded and beads were washed in 2% BSA and centrifuged for 1 min at 14,800g.
6. Alexa-488-tagged secondary antibodies (Life Technologies, 3 ml of antibody in 20 ml of 2 % BSA) were used during 30 min with rotation at 4 ℃. Secondary antibody incubation alone was used as control and to gate the beads with X＋-bound exosomes. The percentage of positive beads was calculated relative to the total number of beads analysed per sample (100,000 events). This percentage was therein referred to as the percentage of beads with X＋ exosomes.
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|Archiver|手机版|外泌体之家 | exosomes & microvesicles
发表于 2015-7-3 11:58:20
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