TITLE: Whole Blood Processing (SepMate)
AUTHORS: Yang Sun, David Lee, Jimmie Ye

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Purpose: To purify PBMCs from 20 or 50mL whole blood (per donor). SepMate tubes will be used with Lymphoprep to ease processing time and effort. </div></div>

[STEPS]
?. [Plasma Separation]
Purify 20mL or 50mL whole blood using the following protocol:
?. [Plasma Separation]
Centrifuge blood samples at swinging-out rotor and brake off within  of collection.
Centrifuge: 1200 34, 10 min
?. [Plasma Separation]
Carefully transfer  from each tube in a 15mL tube avoiding the buffy coat, use remaining blood for PBMC isolation.
[plasma]
Note: To minimize PBMC loss during plasma separation, leave the clear layer level at least 1cm above the RBC.
?. [Plasma Separation]
Centrifuge plasma at  in fixed-angle rotor (if a high-speed centrifuge for 15mL tubes is not available, aliquot plasma in multiple 1.5mL tubes and centrifuge at ). Transfer plasma to new tubes avoiding the pellet.
Centrifuge: 16000 34, 10 min, 4 10
Centrifuge: 16000 34
?. [Plasma Separation]
Aliquot  in multiple 1.5mL low bind tubes, freeze at  on the day of collection and move plasma samples toafter .
[plasma]
-20 °C
-80 °C
?. [PBMC isolation]
Prepare SepMate tubes with Lymphoprep
?. [PBMC isolation]
Dilute whole blood 1:2 with DPBS (Ca/Mg free).
For 105mL total:   and  .
?. [PBMC isolation]
Slowly pipette  down the side of the tube.
[diluted blood]
Some mixing may occur between the medium and blood, but take care not to mix under the divider.Unlike conventional gradient separation, do not tilt the tube when adding diluted blood.
?. [PBMC isolation]
Spin at   with brakes ON.
Centrifuge: 1200 34, 15 min, 20 10
Prepare three destination 50mL tubes (~30mL per Sepmate tube will be yielded.)
?. [PBMC isolation]
When the spin finishes, pour off the top layer from the SepMate tubes into your prepared destination tubes.
Do not invert the tubes for more than 2 seconds.
?. [PBMC isolation]
Prepare 3 SepMate tubes per donor and add  below the insert by pipetting into the center hole.
[Lymphoprep]
Take care to minimize any air bubbles below the plastic divider.Video from SepMate manufacturers (~2min)
?. [PBMC isolation]
Spin at  with brake ON.
Centrifuge: 450 34, 10 min
First wash at higher RCF because gradient medium is mixed with the cells.
?. [PBMC isolation]
Pour off supernatant and combine pellets in three tubes to one, add EasySep buffer to 50mL.
?. [PBMC isolation]
Spin at  with brakes ON.
Centrifuge: 350 34, 10 min
If yield is critical, ensure that the supernatant is clear; opaque supernatant indicates incomplete centrifugation of cells. If necessary, perform the spin at  .
?. [PBMC isolation]
Pour off supernatant, wash in  for a second wash.
[EasySep buffer]
?. [PBMC isolation]
Spin   with brakes on.
Centrifuge: 350 34, 10 min
?. [PBMC isolation]
Pour off supernatant and resuspend in .
[EasySep buffer]
?. [PBMC isolation]
Perform cell count.
?. [PBMC isolation]
Spin at  and resuspend in appropriate volume of freezing media A to get 10e7 cells/mL.
Centrifuge: 350 34, 10 min
?. [PBMC isolation]
Prepare cryovials with .
[freezing media B]
?. [PBMC isolation]
Add  from previous step to cryovials and gently mix by pipetting.
[cell suspension]
?. [PBMC isolation]
Place in freezing container for 1 day at  and move to LN2 tank for long term storage on the following day.
-80 °C
?. [Plasma Separation]
Centrifuge blood samples at swinging-out rotor and brake off within  of collection.
Centrifuge: 1200 34, 10 min
?. [Plasma Separation]
Carefully transfer  from each tube in a 15mL tube avoiding the buffy coat, use remaining blood for PBMC isolation.
[plasma]
Note: To minimize PBMC loss during plasma separation, leave the clear layer level at least 1cm above the RBC.
?. [Plasma Separation]
Centrifuge plasma at  in fixed-angle rotor (if a high-speed centrifuge for 15mL tubes is not available, aliquot plasma in multiple 1.5mL tubes and centrifuge at ). Transfer plasma to new tubes avoiding the pellet.
Centrifuge: 16000 34, 10 min, 4 10
Centrifuge: 16000 34
?. [Plasma Separation]
Aliquot  in multiple 1.5mL low bind tubes, freeze at  on the day of collection and move plasma samples toafter .
[plasma]
-20 °C
-80 °C
?. [Plasma Separation]
Prepare SepMate tubes with Lymphoprep:
?. [Plasma Separation]
Prepare 2 SepMate tubes per donor and add  below the insert by pipetting into the center hole.
[Lymphoprep]
Take care to minimize any air bubbles below the plastic divider.Video from SepMate manufacturers (~2min)
?. [Plasma Separation]
Dilute whole blood 1:2 with DPBS (Ca/Mg free).
For 42mL total:   and  .
?. [Plasma Separation]
Slowly pipette  down the side of the tube.
[diluted blood]
Some mixing may occur between the medium and blood, but take care not to mix under the divider.Unlike conventional gradient separation, do not tilt the tube when adding diluted blood.
?. [Plasma Separation]
Spin at   with brakes ON.
Centrifuge: 1200 34, 15 min, 20 10
Prepare one destination 50mL tube (~25mL per Sepmate tube will be yielded.)
?. [Plasma Separation]
When the spin finishes, pour off the top layer from the SepMate tubes into your prepared destination tubes.
Do not invert the tubes for more than 2 seconds.
?. [Plasma Separation]
Spin at  with brake ON.
Centrifuge: 450 34, 10 min
First wash at higher RCF because gradient medium is mixed with the cells.
?. [Plasma Separation]
Pour off supernatant, add EasySep buffer to 50mL.
Can scale washes to 30mL to save reagents.
?. [Plasma Separation]
Spin at  with brakes ON.
Centrifuge: 350 34, 10 min
If yield is critical, ensure that the supernatant is clear; opaque supernatant indicates incomplete centrifugation of cells. If necessary, perform the spin at  .
?. [Plasma Separation]
Pour off supernatant, wash in  for a second wash.
[EasySep buffer]
?. [Plasma Separation]
Spin   with brakes on.
Centrifuge: 350 34, 10 min
?. [Plasma Separation]
Pour off supernatant and resuspend in .
[EasySep buffer]
?. [Plasma Separation]
Perform cell count.
?. [Plasma Separation]
Spin at  and resuspend in appropriate volume of freezing media A to get 10e7 cells/mL.
Centrifuge: 350 34, 10 min
?. [Plasma Separation]
Prepare cryovials with .
[freezing media B]
?. [Plasma Separation]
Add  from previous step to cryovials and gently mix by pipetting.
[cell suspension]
?. [Plasma Separation]
Place in freezing container for 1 day at  and move to LN2 tank for long term storage on the following day.
-80 °C