Coating beads with photoreceptor outer segments (POS)
Department of Physics, Willamette University
Coating beads with photoreceptor outer segments (POS)
POS fragments are stored as 100 uL aliquots in the -80. In preparation for the protocol: bring the sterile 0.9% NaCl solution (which is in a large, 500 mL container in the refrigerator) into the sterile tissue culture hood, and pipet 2 mL of the solution into a 15 mL conical. This will be our non-sterile stock for the day (this is more than enough for the protocol). Return the NaCl to the refrigerator.
PROTOCOL
1) Wash 35 uL of 2.0-um-diameter beads 3x in PBS. You will only need 21 uL for the POS beads; save the other cleaned beads for later steps in the protocol.
- These are 2.01-um diameter silica spheres from Bangs Labs in the lab refrigerator, make sure to vortex before pipetting 35 uL of beads
- Use 1x non-sterile PBS in the fridge, which is stored in a 15-mL conical
- Protocol for washing beads:
- a) spin down 1minute in our lab centrifuge, remove supernatant, re-suspend in PBS
- b) repeat two more times
2) Make POS/bead mixture in diwater (in 1.5 mL eppendorf tubes)
- RECIPE: 30 uL of POS + 21 uL of beads + 249 uL of diwater
- POS is found in the -80-freezer, get fresh diwater from the lab
3) Agitate for 1 hour by spinning for 1 hour on a rotary wheel at 4 C in the dark
4) Centrifuge for 20 min at 6600 rpm at 4 C (biology refrigerated benchtop centrifuges)
5) Remove supernatant, re-suspend pellet in 300 uL of 0.9% NaCl solution (Wash 1)
6) Centrifuge for 20 min at 6600 rpm at 4 C
7) Remove supernatant, re-suspend pellet in 300 uL of 0.9% NaCl solution (Wash 2)
9) Centrifuge for 20 min at 6600 rpm at 4 C
10) Remove supernatant, re-suspend pellet in 300 uL of 0.9% NaCl solution (Final stock)
FOR BEAD INTERNALIZATION: Add 25 uL of the 300 uL final volume directly to the well with the coverslip. Rock the chamber to mix well. Incubate for 30 minutes, and then make a sample.
