Work Instruction
WI0002
Rev: B
Preparation of Neutrophils from Whole Blood for Selectin Adhesion Assay
1. Materials
1.1. Name: Heparin [1,000 USP Units/mL]
1.1.1. Quantity: 10 Units per mL blood [100L per 10mL Blood]
1.1.2. Manufacturer/Supplier: American Pharmaceutical Partners, Inc.
1.1.3. Catalog Number: 401586
1.2. Name: Latex Free 1 cc U-100 Insulin Syringe
1.2.1. Quantity: 1
1.2.2. Manufacturer/Supplier: Becton Dickinson
1.2.3. Catalog Number: 329420
1.3. Name: Latex Free 60 mL Syringe
1.3.1. Quantity: 1
1.3.2. Manufacturer/Supplier: Becton Dickinson
1.3.3. Catalog Number: 309653
1.4. Name:Vacutainer Blood Collection Set (21 Gauge)
1.4.1. Quantity: 1
1.4.2. Manufacturer/Supplier: Becton Dickinson
1.4.3. Catalog Number: 367281
1.5. Name: 1-Step Polymorphs
1.5.1. Quantity: 3.5 mL per 3.5-5mL Whole Blood
1.5.2. Manufacturer/Supplier: Accurate Chemical & Scientific Corporation
1.6. Name: Hank’s Balanced Salt Solution, .1m filtered without Calcium or
Magnesium, Supplemented with 10mM HEPES (buffer)
1.6.1. Quantity: Varies
1.6.2. Manufacturer/Supplier: Gibco
1.6.3. Catalog Number: 14175-095
1.7. Name: 1 M CaCl2 Stock Solution
1.7.1. Quantity: 20 L per 10 mL Assay Solution (2 mM Assay CaCl2)
1.8. Name: 15 mL Centrifuge Tubes
1.8.1. Quantity: 1 per 3.5-5 mL Whole Blood
1.8.2. Manufacturer/Supplier: Grenier Bio-One
1.8.3. Catalog Number: 188271
1.9. Name: PBS without Calcium or Magnesium
1.9.1. Quantity: Varies
1.9.2. Manufacturer/Supplier: BioWhittaker
1.9.3. Catalog Number: 17-516F
1.10.
Name: .5 M EDTA Stock Solution
1.10.1. Quantity: Varies
1.11.
Name: RPMI Medium 1640
1.11.1. Quantity: Varies
1.11.2. Manufacturer/Supplier: Invitrogen
1.11.3. Catalog Number: 11875-093
2. Procedure
2.1. Blood Collection (MUST BE TRAINED!)
bme.virginia.edu/lawrence
Date: 9/01/07
1/2
Work Instruction
WI0002
Rev: B
Preparation of Neutrophils from Whole Blood for Selectin Adhesion Assay
2.1.1. Place enough Heparin in 60 mL syringe for quantity of blood to be drawn
(refer to 1.1.1) using the insulin syringe. Each small division on insulin
syringe is 20 L.
2.1.2. Clean skin with alcohol wipe, fully dry before proceeding.
2.1.3. Attach the Vacutainer Blood Collection Set to the 60 mL syringe and draw
blood, puncturing skin with bevel up. Insert towards heart to avoid valve
damage.
2.2. Neutrophil Isolation
2.2.1. Prepare 3.5 mL of Polymorphs in 15 mL centrifuge tubes, 1 centrifuge
tube for every 5 mL of blood.
2.2.2. Slowly add whole blood (3-5mL) to top of Polymorphs.
2.2.3. Centrifuge at 500Xg for 50 min.
2.2.4. Draw off bottom layer of white cells (PMNs) and combine in a 50 mL
centrifuge tube.
2.2.5. Dilute 1:1 with PBS solution.
2.2.6. Centrifuge at 400Xg for 10 min.
2.2.7. Draw off the supernatant and resuspend the pellet in PBS, diluting to
initial volume when neutrophils were removed from polymorphs.
2.3. Cell Counting and Assay Media Preparation
2.3.1. Dilute 100 L of neutrophil fluid to 1 mL with PBS.
2.3.2. Inject ~12 L into cell counting plate (until plate is coated)
2.3.3. Count neutrophils in two 4X4 corner cells and average: this yields the
concentration in averageX104 cells/mL in the dilution, or averageX105 in
the neutrophil fluid.
2.3.4. To create 10mL of 1X105 cells/mL with 2 mM CaCl2:
2.3.4.1. Calculate the volume of neutrophil solution (not diluted) required:
2.3.4.1.1. Volume (mL) = 106/(Average*105)
2.3.4.2. Dilute to 10 mL with 5 mL HBSS and 5 mL RPMI.
2.3.4.3. Add 20 L of 1 M CaCl2.
2.3.5. Prepare 1% EDTA Solution in HBSS by diluting .5 mL of stock to 50 mL
with HBSS.
3. Revision History
Date
Rev Description
1/05/04 Initial release
2/07/07 A
Updated to include RPMI in leukocyte flow
buffer.
9/10/07 B
Changed heparin to 10 U/mL. Updated
format.
bme.virginia.edu/lawrence
Date: 9/01/07
Revision Author
Brian J. Schmidt
Brian J. Schmidt
Brian J. Schmidt
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