Sorting TECs from Non-TECs

Contents:

 

 

 

CD45 Depletion

  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 1, 2, 3, 4 and 5. (1500 X 5min X 4 ° C)
  2. Remove and discard supernatant
  3. Add FACS buffer and CD45 beads (Bead Fr1+2 separately from Fr3-5)
  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 in duplicate “+” and “-“ for each sample
  4. Run “Depletes” program on Automacs
  5. Do “Rinse” between samples and “QRinse between duplicates.

 

  1. Discard “+” after 2 nd run
  2. Pool duplicate “-“
  3. Make “-“ up to 10mL with FACS buffer
  4. Spin at 1500 for 5min at 4 ° C and resuspend in 2mL buffer
  5. Count cells

 

 

Sort Sample Staining

Sort sample staining: - can stain samples still in yellow tubes.

10uL per 106 cells

    1. Add epcam to samples
    2. Incubate for 15min at 4 ° C, wash and spin
    3. Add a Rat FITC to samples
    4. Incubate for 15min at 4 ° C, wash and spin
    5. Add 5uL NRS and incubate for 5min
    6. Add CD45Percp-Cy5.5/MHCII
    7. Incubate for 15min at 4 ° C and spin
    8. Remove supernatant, resuspend 300-400uL FACS buffer transfer to sorting tubes (5mL round bottom capped tubes)

Sort comps:

§       Cells alone

§       Epcam + a -rat FITC

§       CD45 FITC

§       MHCII PE

§       CD45 Percp Cy5.5

FACS Antibodies

 

 

Primary

Secondary

Controls

CA

 

 

 

CD45.2 FITC

CD45.2 FITC

 

 

MHCII PE

MHCII PE

 

 

G8.8a BIO/SA-PeCy5

G8.8a BIO

SA-PeCy5

 

CD45 PeCy5

CD45 PeCy5

 

 

UEA-1 FITC

UEA-1 FITC

 

 

6C3 BIO/SA-APC

6C3 BIO

SA-APC

 

6C3 + MHCII PE + CD45 PeCy5

6C3 FITC

MHCII PE

CD45 PeCy5

SA-APC

 

Isotype + MHCII PE + CD45 PeCy5 + UEA-1 FITC

Isotype BIO

MHCII PE

CD45 PeCy5

UEA-1 FITC

SA-APC

 

G8.8a BIO

G8.8a BIO

 

 

Isotype BIO

Isotype BIO

 

 

CD45 FITC + MHCII PE

CD45 FITC

MHCII PE

SA-PeCy5

 

6C3 BIO

6C3 BIO

 

 

UEA-1 FITC + MHCII PE + CD45 PeCy5/SA-APC

UEA-1 FITC

MHCII PE

CD45 PeCy5

SA-APC

Samples

Pre Bead NY

G8.8a BIO

CD45 FITC

MHCII PE

SA-PeCy5

DAPI

 

Pre Bead NA

 

 

 

Post Bead NY

Isotype BIO

CD45 FITC

MHCII PE

SA-PeCy5

DAPI

 

Post Bead NA

 

 

 

Sort Sample NY

G8.8a BIO

CD45 FITC

MHCII PE

SA-PeCy5

DAPI

 

Sort Sample NA