Shreffler:PBMC Isolation

Equipment and Supplies

• Beckman Coulter (Allegra™ X-12R) centrifuge
• Hemacytometer (Reichert Bright line). Fisher # 02-671-5
• Nalgene Cryo Freezing Container filled with 2-propanol. Fisher #15350-50
• – 80° C Freezer
• Biological safety cabinet. Forma Scientific Model 1286 or comparable vendor
• Sterile hood-bench top

• Blood collection and basic lab supplies. Gloves, tube racks, sharps containers, microscope, Kimwipes, ethanol squirt bottle, cell counter, calculator, pen, paper, beaker for decanting, pipet aid, balance, boxes to hold cryovials, timer
• Sterile 50 mL conical tubes. Fisher #14-432-22
• Sterile 15 mL conical tubes. Fisher #14-959-53A
• Ficoll Paque Plus [density 1.077 g/mL, endotoxin tested < 0.12 EU/mL] (Amersham Biosciences; StemCell Technologies)
• 50 mL Accuspin tubes, radiation sterilized. Sigma #A-2055
• 12 mL Accuspin tubes, radiation sterilized. Sigma #A-1805
• Sterile, graduated transfer pipets. Fisher # 13-711-20
• 1.8 mL sterile cryogenic vials. Fisher # 12-565-171N
• 5 mL snap top tube (BD Falcon 12 x 75 mm) for RAST sample. Fisher #14-959-11A (Falcon # 352063)
• Sterile Dulbecco’s Phosphate buffered saline (PBS), Ca++ and Mg++ free. VWR#45000-436
• RPMI – 1640 plus antibiotics (Expires 1 month after preparing)
• AIM V Media (w/HAS). Invitrogen # 12055-091 (Expires 1 month after opening)
• Freezing Medium A. Tissue culture tested, heat inactivated, filtered, Human AB serum – supplied by Univ WI
• Freezing Medium B. 20% DMSO in Freezing Medium A – Supplied by Univ WI
• Sterile 100 ul tips. Fisher # 21-381-8a.
• Non-sterile tips. Fisher # 21-197-8g
• Sterile Pasteur pipet (9”)
• Pipet, 10-100 ul. Fisher # 05-402-88
• Turk’s solution. VWR Catalog: #RC885016 – 500mL, #RC885032 – 1L
• Microcentrifuge tubes. Fisher # 02-681-290
• Serological pipet, 25 mL. Fisher # 13-678-14b