CRC Tissue Core Cytogenetics Studies, SOPs
Mayo Cytogenetics
CpG SOP
Dr. Daniel Van Dyke
Stephanie Smoley
4/20/07
Cytogenetics Laboratory Technical Procedure Manual
Cytogenetics Home
Procedure Manual
Reagents
Stephanie Smoley, Development
AUTHOR:
Technologist
Matthew Bloxham, Culture
Technologist
Mark Law, Research Technologist
APPROVING
AUTHORITY
(IES):
EFFECTIVE
DATE:
Dr. Daniel Van Dyke, Co-director,
Cytogenetics Laboratory
Doc:
Version: 001
10/4/2006
Mayo Clinic - Department of Laboratory Medicine and Pathology
Rochester, MN 55905
B-CELL CULTURES WITH CpG OLIGONUCLEOTIDE FOR CHRONIC
LYMPHOCYTIC LEUKEMIA
PURPOSE
This culture method stimulates B-CLL cells to enter mitosis in >80% of patients. This
culture has been successful for blood and lymphoid tissue, but should also work for bone
marrow specimens.
PRINCIPLE
This test should be ordered by physicians or clients for patients who are suspected of
having B-cell chronic lymphocytic leukemia.
SPECIMENS
Bone Marrow
Draw 1 to 2 mL of unclotted bone marrow in a sodium heparin vacutainer.
Peripheral Blood
Draw 6 mL of unclotted peripheral blood in a sodium heparin vacutainer.
Lymphoid Tissue
At least 50 mg of lymphoid tissue. (spleen, tonsil, lymph node, etc)
REAGENTS / SUPPLIES (See Master Reagent/Supplies Doc. 6500)
CAUTION: All reagents are potentially hazardous. Use appropriate safety procedures
when handling these materials. Avoid contact with skin and mucous membranes. For
more information see the sections pertaining to safety in the laboratory procedures
manual, the Material Safety Data Sheets (MSDS), the Mayo Clinic Safety Manual, and
the Mayo Foundation Chemical Hygiene Plan.
EQUIPMENT
Laminar Flow Hood
Vortexer (Thermolyne Type 16700 Mixer)
Centrifuge
Oxford Pipettor
Thermotron Slide Drying Chamber
Erlenmeyer Vacuum Flask
Incubator/gas processor: Forma
Eppendorf repeater pipettor
QUALITY CONTROL
- The following steps are followed for centrifuge harvested specimens:
1. Aspirate slowly at the top of the meniscus.
2. Avoid aspirating too close to the cell pellet. No less than 1 mL above.
3. Change pipets or aspirate about 1 mL of methanol between each patient after
hypo/prefix change.
4. Change vacuum aspiration flask when ½ full.
5. Remove pipet of vacuum flask when not in use or when shut off.
6. Change vacuum filters weekly (if necessary).
7. Gently add reagents (fix and hypo) to centrifuge tube to avoid splashing and do not put
pipettor tip directly into centrifuge tube while adding reagent.
8. If you suspect cross contamination at any stage of the harvest procedure, notify the
lead technologist and the supervisor.
- In addition to matching patient name and lab I.D. number, bar coding is also performed
between each transfer step (i.e. specimen vial to culture flask, culture flask to centrifuge
tube and centrifuge tube to microscope slides).
PROCEDURE
Culture Procedure
1. Label the flask with the labels provided by MGS indicating the patient name,
accession number, Lab ID, date, specimen type, and culture designation (CpG 5
Day).
2. To assure match, bar code flasks and sample label.
3. Add 0.5 mL of whole bone marrow or blood, or amount of specimen according to
cell count, to a flask containing 5 ml of CpG media. See Use of Coulter AcT
method and see table below.
4. If using lymphoid tissue, push lymphoid tissue through a sterile screen into a Petri
dish to produce a cell suspension.
5. Add the proper number of cells from the resulting cell suspension according to its
cell count to a flask containing 5 ml of CpG media. See Use of Coulter AcT
method and see table below.
6. Cap tightly and mix the culture by gently swirling.
7. The flasks are placed on the flat side and incubated at 37° C with 5% CO2 , 5% O2
and 90% N2.
8. Harvest the specimen after 5 or 6 days in culture.
Total WBC
<5x103/µL
5-9x103/µL
10-49x103/µL
>50x103/µL
Plant into a 5 mL culture
1.5 mL
1.0 mL
0.5 mL (500 µL)
0.25 mL (250 µL)
*Note: If Coulter AcT is not available, plant 0.5 mL of specimen in 5 mL of medium.
Harvest Procedure
1. The day before harvest, add 50 µL of colcemid working solution (1µg/1mL) to
each culture before 5:00 pm.
2. After the colcemid is added, cap the culture flask tightly and mix by gently
swirling.
3. The flask is placed on the flat side and incubated at 37°C with 5% CO2, 5% O2
and 90% N2.
4. After the culture has incubated overnight, prepare a 15 mL screw capped conical
centrifuge tube by labeling it with a patient identification label provided by MGS.
5. Carefully read the patient name and Lab ID numbers on the flask and the
centrifuge tube and make sure they match. Also, bar code flask and centrifuge
tube to assure match. Gently swirl the culture and then pour the contents into the
centrifuge tube.
6. Recap the centrifuge tube and centrifuge at 1,200 rpm for 8 minutes.
7. Aspirate all but 1 mL of supernatant using the Erlenmeyer vacuum flask.
8. While gently vortexing, add 10 mL of potassium chloride hypotonic solution
(0.075M).
9. Cap the centrifuge tube and incubate for 30 minutes at room temperature.
10. Add 2 mL of fixative (2:1), cap centrifuge tube tightly and mix by gently
inverting the centrifuge tube 2 or 3 times.
11. Centrifuge at 1,200 rpm for 8 minutes.
12. Using the Erlenmeyer vacuum flask, aspirate all but 1 mL of supernatant,
changing pipets or aspirating 1 mL of methanol between each pipette.
13. While gently vortexing, add 10 mL of fixative (2:1) using a serological pipet
(10mL).
14. Recap the centrifuge tube and resuspend the cells again by inverting the tube 2 to
3 times.
15. Centrifuge at 1,200 rpm for 8 minutes.
16. Using the Erlenmeyer vacuum flask, aspirate all but 1 mL of supernatant.
17. Resuspend the cell suspension by gently vortexing. Add 10 mL of fixative (2:1)
using an Oxford pipettor.
18. Recap the centrifuge tube and centrifuge at 1,200 for 8 minutes.
19. Repeat steps 16 to18 at least two more times. If the cell pellet is dirty, continue
with steps 16 to 18 until the cell pellet becomes clear.
Slide preparation
1. For slide preparation see: Slide Preparation Using the Thermotron Slide Drying
Chamber.
Literature cited:
1.
2.
Decker, T., et al., Immunostimulatory CpG-oligonucleotides cause proliferation,
cytokine production, and an immunogenic phenotype in chronic lymphocytic
leukemia B cells. Blood, 2000. 95(3): p. 999-1006.
Decker, T., et al., Immunostimulatory CpG-oligonucleotides induce functional
high affinity IL-2 receptors on B-CLL cells: costimulation with IL-2 results in a
highly immunogenic phenotype. Exp Hematol, 2000. 28(5): p. 558-68.
REAGENTS
BSA working solution (10%): Add 10 g BSA (Seriologicals, cat# 3225-80) to 100 mL
PBS in volumetric flask. Freeze at -20°C. Use within 1 year.
Colcemid stock solution (10 µg/mL): 0.001 g colcemid (SIGMA Chemical Co. 50 mg
lots) in 100 mL of Hanks balanced salt solution (1X)(Irvine Scientific). Sterilize by
filtration. Refrigerate at 2o to 8o C. Use within 1 year.
Colcemid working solution (1 µg/mL): 90 mL of Hanks balanced salt solution (1X)
(Irvine Scientific) and 10 mL colcemid stock solution (10 µg/mL). Sterilize by filtration.
Refrigerate at 2o to 8o C. Use within 1 year.
CpG stock solution: 200µg (25.96 nmol) lyophilized ODN2006 (ODN-B) (InvivoGen,
cat# tlrl-hodnb, ODN 2006 sequence) in 2mL sterile endotoxin-free water.
Fixative (2:1): 20 mL methanol (Methanol anhydrous (absolute) acetone free) and 10
mL glacial acetic acid (Reagent grade, meets ACS specifications). Prepare fresh daily.
Interleukin-2 (IL-2) stock solution: Resuspend 10µg IL-2 (PeproTech Inc., cat# 20002) in 40µl of 100mM acetic acid to reach a final concentration of 2.5x106 U/ml. Store at
-20°C. Use within 1 year.
Interleukin-15 (IL-15) stock solution: Add 20µL of 10% BSA solution to 2mL of PBS.
Add 1ml BSA/PBS solution to 10µg IL-15 (PeproTech Inc., cat# 200-15) to reach a final
concentration of 10µg/ml. Store at -20°C. Use within 1 year.
Mercaptoethanol (2-ME) working solution: Add 37µL of ME (Sigma, cat# M-6250)
to 1mL of PBS. Add 10µL of this solution to 1mL of PBS.
Penicillin, streptomycin solution (penstrep): Invitrogen. Cat. No. 15140-122. 10,000
units/mL penicillin G, 10,000 µg/mL streptomycin sulfate. 100 mL lots (frozen). Store at
-20°C in 2.5 mL lots for 2-100 mL medium, and 10 mL lots for 2-500 mL medium. Use
within 6 months.
Potassium chloride hypotonic solution (0.075 M): 11.18 g KCl and bring up to
2,000 mL with deionized water. Check with pH indicator strip (~7.0 pH). Use within 1
year. Prepared by Preparation and Processing laboratory. Hilton 552.
RPMI 1640 medium with GlutaMAX: Invitrogen Corp. Cat. No. 61870-036. 500 mL
lots. Refrigerate. Protect from light. Use within 1 year.
CPG media is made by adding the following reagents:
 180mL of RPMI Glut Max media
 10mL BSA working solution
 200µL of ME working solution
 2mL of penstrep
 12mL of CPG stock solution
 200µL of IL-15 stock solution
 2µL of IL-2 stock solution
Filter using 0.22 micron filter. Add 5mL to T-25 unvented tissue culture flasks. Store at
-20 C.