CD45 Depletion (Thymus)

Contents:

 

 

 

Suspension/Digestion

  1. Clean thymus
  2. Put in RPMI buffer (yellow capped tube)
  3. Cut 3 – 4 times and digest with snapped off glass pipette (filter in top)
  4. Let settle

 

  1. Remove RPMI and put in blue capped tube = THYMOCYTES
  2. Add ~5mL RPMI
  3. Agitate
  4. Let settle

Repeat steps 5 – 8 until solution becomes clear

  1.   Add 5mL Enzyme mix (4mL collagenase, 1mL DNase) to each tube
  2. Agitate with wide ball pipette
  3. 37 ° C water bath for 10 minutes
  4. Agitate
  5. Water bath 10 minutes
  6. Let settle
  7. Remove supernatant = FRACTION 1

 

  1. Add 5mL enzyme mix to each tube
  2. Agitate with wide ball pipette
  3. 37 ° C water bath for 10 minutes
  4. Agitate
  5. Water bath 10 minutes
  6. Agitate
  7. Let settle
  8. Remove supernatant = FRACTION 2

 

  1. Add 5mL enzyme mix to each tube
  2. Agitate with wide ball pipette
  3. Water bath 10 minutes
  4. Agitate with skinny glass pipette
  5. Water bath 10 minutes
  6. Agitate
  7. Let Settle
  8. Remove supernatant = FRACTION 3

 

  1. Add 5mL enzyme mix to each tube
  2. Agitate with skinny pipette
  3. Water bath 10 minutes
  4. Agitate
  5. Water bath 10 minutes
  6. Agitate
  7. If broken up enough, let settle and remove supernatant = FRACTION 4

 

  1. Add 5mL Enzyme mix to each tube
  2. Agitate with blue pipette
  3. Water bath 5 minutes
  4. Agitate
  5. Water bath 5 minutes
  6. Agitate
  7. Make up to 10mL with RPMI
  8. Filter into yellow capped tube
  9. Do counts of each fraction

 

  1. Spin down fractions 3, 4 and 5. (1500 X 5min X 4 ° C)
  2. Remove and discard supernatant
  3. Add FACS buffer and CD45 beads
  4. Leave in cool room, mixing, for 20 minutes (2 1/2 speed)
  5. Top up to 10mL with FACS buffer
  6. Spin at 1200 for 10 minutes at 4 ° C (Run “Clean” on Automacs while spinning)

 

  1. Discard supernatant
  2. Resuspend in 6 – 8mL of FACS buffer (depending on how “thick” the solution is)
  3. Label tubes “+” and “-“ for each sample
  4. Run “Depl025” program on Automacs
  5. Do “Rinse” between samples.

 

  1. Discard “+” after run
  2. Make “-“ up to 10mL with FACS buffer
  3. Spin at 1500 for 5min at 4 ° C and resuspend in 2mL buffer
  4. Count cells
  5. Spin at 1500 for 5 min at 4 ° C
  6. Resuspend in 350uL RLT buffer and snap-freeze. Store at -80 ° C
  7. Make up cDNA

 

FACS Analysis

  • Stain with CD45.2 FITC
      • Cells alone
      • CD45.2 FITC comp
      • Pre sort (1x10 6 cells from combined fractions)
      • Post sort (20uL from 2mL)

Buffers

FACS Buffer:

    • 5mL EDTA
    • 5mL 10% BSA
    • 490mL 1XPBS

Collagenase: 60mg in 40mL RPMI

DNAse: 10mg in 10mL RPMI