Blood Processing Procedure

Red Blood Cell Membrane preparation process1. Warm hands in hot water and dry. Obtain finger-stick blood sample (Lab assistants will do this).2. Mix the collection syringe (no needle) to mix in anticoagulant and gently transfer the blood to a1.5 mL snap top conical tube, rinse the syringe with osmotically balanced 0.15M NaCl solutionand write your initials on the tube.3. Centrifuge your conical tube for 1 minute on highest setting to pellet the red blood cells andleave the major portion of the yellow plasma on top.4. Using a fresh pipet, remove the yellow plasma top layer from conical tube and discard intobleach bucket. This top plasma layer may also be refereed to as the supernatant (a liquidoverlaying a solid).Note: This will leave you with a small red cell pellet at the bottom of your conical tube.5. Use a fresh plastic pipet and add 0.15 M NaCl (sodium chloride) up to the 1.5 mL line on yourconical tube.6. Vortex the conical tube for 10 seconds to resuspend the red blood cells.7. Centrifuge your conical tube for 1 minute on highest setting to repellet the red cells and washoff plasma that was between the red cells in the initial red cell pellet..8. Using a fresh pipet, remove the top layer (plasma/supernatant wash) from conical tube anddiscard pipet into bleach bucket.Note: Again, this will leave you with a small red cell pellet at the bottom of your conical tube.9. Add deionized (DI) water using a fresh pipet to your conical tube containing the red blood cell(RBC) pellet up to the 1.5mL mark.Note: This step and the next will lyse (burst) the red blood cells.10. Set vortex to max and vortex conical tube for 10 seconds to resuspend the red cells andencourage their bursting.11. Set centrifuge to max and centrifuge conical tube for 5 minutes. (This step will create a pellet ofRBC ghost membranes.)Note: The RBC ghost membrane pellet is very small and may be hard to observe.12. Obtain a fresh plastic pipet and remove the red hemoglobin supernatant by slowly pipetingdown to the 0.3 mL mark on your conical tube. Discard the red hemoglobin supernatant into thebleach bucket.Note: Be careful not to remove all of the red hemoglobin supernatant as you may loose your RBCmembranes.Now you have obtained your RBC membranes and are ready to test them to determine your red bloodcell fatty acid composition and the omega 3/omega 6 fatty acid ratio. You will conduct the next portionof the lab with the help of a teaching assistant please wait for their assistance before continuing.

Extraction of RBC lipids & preparation of volatile fatty acid methyl esters1. With help of TAs, use Pipetman pipette (automatic pipet) to add 1.0 mL of 2:1 DCM(dichloromethane):methanol solution, containing a known amount of C15:0 fatty acid internalstandard, to the conical tube containing your RBC membranes and cap tightly.Note: This step will extract the fatty lipids from the RBC membranes.2. Set vortex to maximum and vortex conical tube for 10 seconds to resuspend the red cellmembrane pellet.3. Set centrifuge to highest setting and centrifuge conical tube for 2 minutes to separate the DCMrich and water rich layers.4. Use Pipetman (with help from TAs) to remove lower clear liquid layer from conical tube andtransfer it to a small glass vial with your initials on vial. Dispose of conical tube.Note: The lower layer (DCM layer) contains the fatty lipids.5. Dry sample in glass vial with nitrogen gas in the organic chemistry lab down the hall. Slowlyand gently start nitrogen flow over sample and let stand for 10-15 minutes or until completelydry.Note: This part of procedure is conducted under a ventilation hood in the organic chemistry lab to pullthe DCM and methanol solvent out of the lab.6. TAs will add 340 uL of 10% BF 3 (Boron Trifluoride) in MeOH (methanol) using Pipetman. Setvortex to maximum and vortex sample for 10 seconds.7. Check to make sure cap is tightly on glass vial containing sample. Place vial in oven set at 60°Cfor 15 minutes.8. Remove glass vial from oven (will be warm) and cool samples in cold water for 10 seconds.Note: Cooling sample prevents MeOH from boiling when cap is opened.9. Add 200 uL of deionized (DI) water to vial using Pipetman.Note: The DI water will kill off any excess BF 3 .10. Set vortex to maximum and vortex sample for 10 seconds.11. Use a Pipetman to add 200 uL of hexane to glass vial. Cap vial tightly. Set vortex to maximumand vortex sample for 10 seconds.12. Let glass vial sit undisturbed for 2 minutes.Note: The liquid in the vial will separate into two layers.13. Remove 50 uL of hexane (top liquid layer) using Pipetman and transfer to glass insert in a newglass sample vial. Initialize new glass vial and dispose of old.14. DONE: TAs will measure the fatty acids in your sample using the Gas Chromatographymachine and report the results to you when you return to MSU for the final parts of this unit.