Peripheral Blood Separation CJF

• 30 mL peripheral blood drawn in heparin tubes (green tops) requested from AM in the blood bank. Request need to be made as early as possible, with a minimun of 24-48hrs lead time. This is requested via email with a reference to the PI. Blood is picked up in the blood bank, specimen pick up area with login. First draws are at 7:30, however research blood could also be drawn at the 11am group.

• “ I would like to request 30ml from a normal volunteer in green tops for ___ date pick up as early as possible. The PI is ____” AM does not work on Friday.

Isolating PBMCs and Neutrophils

Sterile techniques and bioharzard precautions at all times

1. Combine blood into a 50mL falcon and dilute 1:1 with cold 1x HBSS without Ca/Mg. 2x50mL falcons, cold 1xHBSS w/o Ca/Mg, 10mL pipet [‘Eyeball’ final volume and add this volume of HBSS without Ca/Mg into another 50mL Falcon. You are going to wash all green tops with HBSS to ensure maximum blood collection and then add this to falcon containing blood. Throughly mix blood by inverting 2-3 times]
2. Overlay blood onto 15mL cold Lymphocyte separation medium without disrupting the layer interface quickly to prevent sedimentation of samples. Cold LSM, 2x50ml Falcon tubes, 25mL pipet (Lymphocyte separation medium is a sterile iso-osmotic polysucrose and diatrozle solution with low viscosity to create a continous gradient post centrifugation allowing the easy separation of Lymphocytes. Mononuclear cells will be contained in the banded Plasma-LSM interphase. PMNs (neutrophils) will be at the banded LSM-RBC layer. LSM contains 106.91 g/L of Diatrizoic Acid and 68.181 g/L Polysucrose 400 at a density of 1.077-1.080 g/mL @ 20 C. The solution has an osmolarity of 290 ± 20 mOsm and a pH of 7.0±2.0. Sodium Hydroxide is added as needed to adjust pH.) [2x 50mL Falcons with 15mL LSM per 30mL blood draw. Tilt the LSM to 45degrees in a stable setting. Slowly add blood:HBSS mix down side of tube to prevent disruption of the layer. Once a thick layer of blood has been created, can speed up to add rest of the blood. Add 30mL of blood:HBSS solution to each 50mL falcon containing 15mL LSM so that the 2 50mL falcons have the same final volume. Cap and quickly proceed to centrifugation step. Delay will cause sedimentation of blood:HBSS into LSM and cause issues with final separation.
3. Centrifuge tubes at 4C, 2,000 rpm, 30min, slow deceleration. [Make sure brake is off and centrifuge is pre-cooled.] Prep for next steps.

Separation of PBMCs

1. Carefully remove Plasma:LSM layer 3mm on each side of layer and place in a new 50 mL falcon tube. [insert 10mL pipet to 3mm above plasma:LSM banded layer and extract layer while slowly moving pipet around and maintaining the falcon tube perfectly still and perpendicular to benchtop. Extract through 3mm below plasma:LSM layer. End extracted volume should be approximately 15mL.]
2. Add cold 1xHBSS without Ca/Mg to final volume of 50mL. [Keep samples separate at this point. Therefore add 15mL PBMCs from each gradient to a 50 mL falcon and add cold 1x HBSS without Ca/Mg.]
3. Spin at 4C, 2,000rpm, 10minutes, fast deceleration. [Brake on] (This removes the LSM)
4. Resuspend and add 15mL cold ACK lysis buffer, mix thoroughly, and place at 4C for 10minutes. (ACK lysis buffer is ammonium-chloride-potassium hypotonic solution that preferientially lysises RBCs) [Resuspend via striking or vortexing]
5. Dilute with cold 1x HBSS without Ca/Mg. [Bring volume of each falcon up to 50mL]
6. Spin at 4C, 2,000rpm, 10min, fast decelartion. [Brake on. Remove supernatant.]
7. Combine samples from patient and resuspend in cold 50mL 1xHBSS without Ca/Mg. [After decanting supernatant from last spin, strike to resuspend cells. Add 25mL HBSS to 1 tube wash sides and vortex tube and add to other tube and ensure cells are resuspended. Do second rinse of 1st tube with additional 25mL cold HBSS and add to 2nd tube.]
8. Spin at 4C, 2,000rpm, 10min, fast decelartion. [Brake on. Remove supernatant.]
9. Resuspend PBMCs in 5.5mL cold media. [Strike tube to resuspend cells and add 5.5mL RPMI 1640 media supplemented with 10-20% HI FBS, 2x HQ HEPES, 1x Glutamine (and or 1x Pen-Strep : depending on next steps). Media is made by filtering 500mL RPMI 1640, 100mL HI FBS, 5 mL 100x glutamine (and or 100x Pen/strep/glutamine), 10ml HQ HEPES. ]