Lidstrom: Flow Cytometer: Difference between revisions

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(New page: Back home: Lidstrom Using the Flow Cytometer 1. Turn on the Flow Cytometer System by flipping the bottom switch on the left side of the machine. 2. Turn on Laser 1 (488 nm) by flipp...)
 
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Back home: [[Lidstrom]]
Back home: [[Lidstrom]]


Using the Flow Cytometer
==Using the Flow Cytometer==
1. Turn on the Flow Cytometer System by flipping the bottom switch on the left side of the machine.
#Turn on the Flow Cytometer System by flipping the bottom switch on the left side of the machine.
2. Turn on Laser 1 (488 nm) by flipping the top switch on the left side of the machine. Laser 2 is controlled through software.
#Turn on Laser 1 (488 nm) by flipping the top switch on the left side of the machine. Laser 2 is controlled through software.
3. Open FloMax Program on the computer (Ugly head Icon)
#Open FloMax Program on the computer (Ugly head Icon)
4. Open Camera (CCD Camera)
#Open Camera (CCD Camera)
5. Flush System
#Flush System
a. In the menu bar, go to “Acquisition,”
##In the menu bar, go to “Acquisition,”
b. Put waste container under the sample insertion tubes
## Put waste container under the sample insertion tubes
c. Select  “Sheath Fluid Prime”
## Select  “Sheath Fluid Prime”
d. Sample insertion tubes should drip
##Sample insertion tubes should drip
e. Flush with 50 mL (~15 min)
## Flush with 50 mL (~15 min)
f. If cell sorting, flush sorting line out with 50 mL (~15 min) too
##If cell sorting, flush sorting line out with 50 mL (~15 min) too
6. Check Camera for bright spots (aka bubbles)
#Check Camera for bright spots (aka bubbles)
a.  Move tubing to try to dislodge bubbles
## Move tubing to try to dislodge bubbles
b. If that fails, dethatch tubing and use syringe to remove bubble
##If that fails, dethatch tubing and use syringe to remove bubble
c. Flush system for 15 minutes after reattaching tubing
## Flush system for 15 minutes after reattaching tubing
7. Open Instrument Setting
#Open Instrument Setting
8. Go to Setup to set a number of cells to be counted by checking the enable button
#Go to Setup to set a number of cells to be counted by checking the enable button
#Hit Clean
# Push sample in cuvette up into chamber
#Push Clean
#Run bead standard (3 uM)
#Clean
#Calibrate with 1,3 and 5 uM beads separately (stored in fridge)
##1 drop of bead solution/1-2 mL
#Repeat for all samples
#Reading the charts
##SSC v. FSC: shows size distribution of the particles
##SSC v. FL1: shows fluorescence separated by size from laser 1
##SSC v. FL2: shows fluorescence separated by size from laser 2
# If needing to break for more than 2 hours, put a cuvette of water in the sample chamber and press end
# After running samples, clean the machine by running the following through the machine
## 1 cuvette of wash solution
## 1 cuvette of 100% ethanol
## 1 cuvette with water
#Put new cuvette of water in the sample chamber
#Close the programs
#Turn off the Laser (top switch on left side)
#Turn off the System (bottom switch on left side)
#Shut down Flomax
 
 
9. Hit Clean
===Media Prep (Should be done the day before the run)===
10. Push sample in cuvette up into chamber
*Media without organic substrates should be used
11. Push Clean
# Filter media
12. Run bead standard (3 uM)
# Autoclave media
13. Clean
# Attach to instrument while media is still hot
14. Calibrate with 1,3 and 5 uM beads separately (stored in fridge)
# Leave overnight
a. 1 drop of bead solution/1-2 mL
15. Repeat for all samples
16. Reading the charts
a. SSC v. FSC: shows size distribution of the particles
b. SSC v. FL1: shows fluorescence separated by size from laser 1
c. SSC v. FL2: shows fluorescence separated by size from laser 2
17. If needing to break for more than 2 hours, put a cuvette of water in the sample chamber and press end
18. After running samples, clean the machine by running the following through the machine
a. 1 cuvette of wash solution
b. 1 cuvette of 100% ethanol
c. 1 cuvette with water
19. Put new cuvette of water in the sample chamber
20. Close the programs
21. Turn off the Laser (top switch on left side)
22. Turn off the System (bottom switch on left side)
23. Shut down Flomax
 
Media Prep (Should be done the day before the run)
Media without organic substrates should be used
1. Filter media
2. Autoclave media
3. Attach to instrument while media is still hot
4. Leave overnight
 
 
OR
OR

Latest revision as of 13:01, 28 June 2013

Back home: Lidstrom

Using the Flow Cytometer

  1. Turn on the Flow Cytometer System by flipping the bottom switch on the left side of the machine.
  2. Turn on Laser 1 (488 nm) by flipping the top switch on the left side of the machine. Laser 2 is controlled through software.
  3. Open FloMax Program on the computer (Ugly head Icon)
  4. Open Camera (CCD Camera)
  5. Flush System
    1. In the menu bar, go to “Acquisition,”
    2. Put waste container under the sample insertion tubes
    3. Select “Sheath Fluid Prime”
    4. Sample insertion tubes should drip
    5. Flush with 50 mL (~15 min)
    6. If cell sorting, flush sorting line out with 50 mL (~15 min) too
  6. Check Camera for bright spots (aka bubbles)
    1. Move tubing to try to dislodge bubbles
    2. If that fails, dethatch tubing and use syringe to remove bubble
    3. Flush system for 15 minutes after reattaching tubing
  7. Open Instrument Setting
  8. Go to Setup to set a number of cells to be counted by checking the enable button
  9. Hit Clean
  10. Push sample in cuvette up into chamber
  11. Push Clean
  12. Run bead standard (3 uM)
  13. Clean
  14. Calibrate with 1,3 and 5 uM beads separately (stored in fridge)
    1. 1 drop of bead solution/1-2 mL
  15. Repeat for all samples
  16. Reading the charts
    1. SSC v. FSC: shows size distribution of the particles
    2. SSC v. FL1: shows fluorescence separated by size from laser 1
    3. SSC v. FL2: shows fluorescence separated by size from laser 2
  17. If needing to break for more than 2 hours, put a cuvette of water in the sample chamber and press end
  18. After running samples, clean the machine by running the following through the machine
    1. 1 cuvette of wash solution
    2. 1 cuvette of 100% ethanol
    3. 1 cuvette with water
  19. Put new cuvette of water in the sample chamber
  20. Close the programs
  21. Turn off the Laser (top switch on left side)
  22. Turn off the System (bottom switch on left side)
  23. Shut down Flomax

 

Media Prep (Should be done the day before the run)

  • Media without organic substrates should be used
  1. Filter media
  2. Autoclave media
  3. Attach to instrument while media is still hot
  4. Leave overnight

  OR If not autoclaving, 1. Flush media with helium for 3-9 hours   Make 20x stock à Appropriate amount of autoclaved water Mix while water is hot and attach to instrument

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