Eric, I have done this type of experiment with 100um beads and it is extremely difficult. I am not the type of person to run away from a challenge but I learned my lesson with this type of sort. When, at the end of the day, you feel totally drained, you are not sure you accomplished much of anything and you have more of these experiments to look forward to running, then you may feel as strongly about this type of experiment as I do. Here are a few points to keep in mind: 1. You can NOT put 180um beads through a 400um nozzle very easily. If just three beads stick together, you will clog the nozzle or at the very least disrupt the droplet formation. Usually the minimum ratio for nozzle to particle size is 4:1. You may be able to get away with a ratio of 3:1 but I think the amount of work is just tremendous. For example, to clear a clog, you will have to increase your sheath pressure, do a nozzle flush, reset the appropriate sheath pressure and continue to sort, hoping that your drop delay has not changed. 2. Just keeping 100um beads in suspension is a huge task. The more you vortex them then more you resuspend bead aggregates and the more clogs you get. Beads will completely settle out in less than 10 minutes. Filtering the beads helps but sometimes they reaggregate. 3. Keeping the bead flow rate constant is difficult because the sample tubing you must use is very large (at least 200um-300um I.D.) and the bead flow rate will be very susceptible to even the slightest changes in sample differential pressure. 4. The beads tend to pile up near the sample injection needle because your sample tubing will have to fit over the outside of the sample injection needle causing a lip to form which stops the beads from flowing into the flow cell. 5. You will go through a lot of sheath fluid. I recommend that at the half day to 3/4 day point, you stop the sort and refill the sheath tank especially if it is the smaller tank. Every time you must stop the sheath, you should let the droplet formation stabilize again. This can take a half hour or so. 6. Since your ddf is 2000-3000 drops/sec your event rate will need to be about 500 beads/sec. Sorting through just 1,000,000 beads is time consuming. 7. I used a sheath pressure of about 2-3 PSI. 8. Use pulse area for your fluorescence measurements. Good luck. Glenn MIT >Our group has been tasked to sort large diameter beads (180 um) which have >gone through a process to attach peptides. We suggested that a new library >of smaller peptide labeled beads be used, but for now the group wants to try >and make use of the larger beads until a new library can be created using >small beads. We have a FACStar Plus equipped with a 400 um nozzle (with >turbo sort option) and we have tried to align/standardize/calibrate with >little success. We have followed the BD macrosort procedure (lower sheath >psi and lower ddf) but again with little success. The only bead that I >currently have for alignment are flow check beads which are ~10 um (I assume >at this point that 10 um beads are not the correct ones to use with the >large nozzle). I have ordered 100 um beads from FCSC. > >- Has anyone sorted using a 400 um nozzle? >- Has anyone successfully sorted ~180 um beads before? >- How do we align/calibrate/standardize the instrument? > >Thank you. > >Eric Nealley >Biologist, Pharmacology Division >Aberdeen Proving Ground, Maryland
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