Measuring Competence : Fixation and Flow Cytometry

Measuring Competence – Fixation and Flow Cytometry The movement of Bio Safety 2 equipment/lab material MUST be well regulated. Since the Flow cytometer is located in Sharpless 305, we need to take extra precautions to prevent contamination via infectious agents when transporting the S. suis samples out of lab. Fixation

1. Centrifuge 1.5 ml epi tube to collect cells and aspirate supernatant

2. Resuspend the pelleted cells in 0.5-1 ml 1X PBS. Add formaldehydepe [FINAL 4%]

3. Allow mixture to sit for 15 minutes at room temperature to fix.

4. Wash via centrifugation with excess 1x PBS. Discard supernatant and resuspend in 0.5-1 ml 1X PBS. Flow Cytometry (Room : S305)

5. Upon Fixation, run the Flow cytometer machine (Refer to the Flow cytometry protocal).

6. Record (a.) Total number of particulates (b.) Total number of red particulates

Protocol adapted from https://www.cellsignal.com/contents/resources-protocols/flow-cytometry-protocol-(flow)/flow