THE UNIVERSITY OF NEWCASTLE- DISCIPLINE OF MEDICAL BIOCHEMISTRY. STANDARD OPERATING PROCEDURE PROCEDURE NO: GLP 146 MOD: 1st Issue Page: 1 of 7

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1 THE UNIVERSITY OF NEWCASTLE- DISCIPLINE OF MEDICAL BIOCHEMISTRY STANDARD OPERATING PROCEDURE PROCEDURE NO: GLP 146 MOD: 1st Issue Page: 1 of 7 Procedure Type: General Laboratory Procedure 1. Risk Assessment: This Risk Assessment is to be used as a general guide and as such, cannot accommodate all the varying factors that may be encountered when using this procedure. Therefore, personnel are requested to conduct their own Risk Assessment before using this procedure to include any extra hazards introduced by the task performed. TASK PERFORMED Harvesting Mononuclear Cells from Human Blood and Bone Marrow HAZARDS 1. Infectious risk human blood and tissue 2. Needle-stick injury 3. RBC Lysis buffer - Irritating to eyes, respiratory system, and, skin. 4. DMSO (Dimethyl Sulfoxide) - Irritant: Slightly hazardous in case of inhalation (lung irritant). Slightly hazardous in case of skin contact (irritant, permeator), of eye contact (irritant). 5. Primocin - May cause eye irritation. May cause skin irritation. May cause sensitization by skin contact. May be harmful if absorbed through the skin. May be harmful if inhaled. May cause respiratory tract irritation. Harmful if swallowed. 6. Penicillin-Streptomycin solution - Irritant. May cause sensitization by skin contact. Irritating to eyes, respiratory system and skin. 7. Wrights-giemsa stain Flammable. Toxic by inhalation, in contact with skin and if swallowed. 8. Vectamount - May cause local discomfort or irritation if placed on skin, in eyes, ingested or inhaled. 9. Liquid nitrogen direct contact may cause injury. RISK ASSESSMENT 1. The risk of handling human blood and tissue is low if routine controls are followed. 2. The risk of needle-stick injury is reduced with the risk controls in place. WRITTEN BY CHECKED BY AUTHORIZED BY NAME (signed) Fiona McDougall Nikki Verrills Deborah Edmunds DATE 7 th Sept th Sept 2011 Distributed To: GLP Master File / GLP Lab File

2 Page: 2 of 7 3. RBC lysis buffer, Primocin, Penicillin-Streptomycin, Wright s-giemsa, Vectamount and DMSO could come into contact with and/or be inhaled by the user. 4. The risk of injury from liquid nitrogen is low if correct procedures are followed. RISK CONTROL 1. Refer to GLP102 Working with human, murine and GM cell lines. 2. Refer to GLP098 Cryopreservation of human, murine and GMO cell lines. 3. Refer to GLP100 Reviving human, murine and GMO cell lines from frozen stocks. 4. Do not resheath needles after use. 5. Dispose of needles directly into sharps container. 6. Wear appropriate personal protective equipment lab gown, safety glasses, and disposable gloves nitrile or vinyl. Ensure adequate ventilation, and work in biohazard cabinet when appropriate. 7. Training should be provided by personnel experienced in this procedure. 8. Training should be undertaken in General Laboratory Safety. 2. Purpose: 2.1. Separate mononuclear white blood cells from blood or bone marrow using the Ficoll method. 3. Equipment: 3.1. Biohazard hood 3.2. Pipettors and pipette boy 3.3. Mr Frosty 3.4. Centrifuge 4. Materials: 1.1 Sterile syringes (2, 5 & 10mL), 21G needles, 18G blunt drawing up needles 1.2 Pipette tips and serological pipettes 1.3 Sterile 15mL and 50 ml centrifuge tubes ml cryovials 1.5 Glass slides and coverslips (22 x 50 mm) 5. Reagents: 5.1. Ficoll-Paque PREMIUM Density (GE Healthcare Product: ) 5.2. Hanks solution or RPMI media 5.3. RBC lysis buffer

3 Page: 3 of Freezing mix IMDM media containing 20% FBS, 10% DMSO and Primocin (1:500 dilution) 5.5. Thaw media IMDM media containing 20% FBS, penicillin-streptomycin (1:50 dilution) and DNase (50 Kunitz U per ml media) note: add DNase just prior to using thaw media. Use SIGMA Cat. No. D Experimental media - media IMDM media containing 0.5% FBS and penicillin-streptomycin (1:50 dilution) 5.7. Wrights giemsa stain 5.8. Vectamount 6. Set Up: 6.1. Ensure that you have read and understood the Safety Precautions (Section 7 of this SOP) prior to commencing this procedure. 7. Safety Precautions: 7.1. Good laboratory techniques are to be used at all times. 8. Method: 8.1. Blood samples - should be provided in an EDTA blood tube (~10mls) In a biohazard hood, mix blood well, and remove lid Take a 20 µl sample & make a blood smear on a glass slide Air dry for a few minutes, then stain using modified Wrights Giemsa (in a coplin jar) for 3 minutes Rinse in distilled water & air dry Mount with Vectamount & a 22 x 50 mm glass cover slip Using a 1ml pipette, transfer the blood to a 50 ml falcon tube, and estimate total volume of blood (eg: 10 ml) Add an equal volume (to blood volume, ie: 10 ml) of Hanks or RPMI media to the original blood tube to flush out all the blood, and transfer to the 50 ml falcon & mix with blood Proceed to step Bone Marrow samples - Bone marrow should be provided in a yellow-top Heparinised RPMI tube (~4mls per tube) If bone marrow/rpmi mixture is clumpy & viscous, spin tubes down for 5 mins at 600rpm, and estimate total volume of bone marrow/rpmi mixture.

4 Page: 4 of In a biohazard hood, using a 1ml pipette, transfer the bone marrow/rpmi mixture to a 50 ml falcon tube Add double the volume (to bone marrow/rpmi mixture, ie: 2x8ml =16 ml) of Hanks or RPMI media to the original bone marrow tubes to flush out all the cells, and transfer to the 50 ml falcon & mix with bone marrow Using a 2 ml syringe & a 18g blunt drawing up needle, draw the bone marrow/rpmi mixture up & down (gently) to break up any cell clumps and blood clots Ficoll protocol for blood and bone marrow: Open the metal tab on the Ficoll bottle, and using a sterile 5 or 10 ml syringe + 21g needle, remove required volume of Ficoll (need 3 ml Ficoll for every 4 mls of blood or bone marrow/hanks mix), and place Ficoll into a 50 ml tube Using a 1 ml pipette, carefully & very slowly, transfer the blood/hanks mixture on top of the Ficoll DO NOT MIX Centrifuge at 400 x g for 40 minutes, at º C Gently draw off the upper layer containing plasma & platelets using a sterile pipette, leaving the layer of mononuclear cells undisturbed at the interface Using a sterile 1 ml pipette, transfer the mononuclear cell layer to a sterile 15 ml tube Add at least 3 x the cell volume of Hanks or RPMI media to the mononuclear cells & suspend the cells gently by mixing up & down with a pipette Centrifuge at x g for 15 minutes at º C, & remove the supernatant For bone marrow samples - if there is excess RBC contamination in the mononuclear cell pellet, add 5-10 mls RBC lysis buffer, wait 5 minutes, and centrifuge for 10 minutes at 800 rpm (20 º C), remove supernatant. Then resume step Add ~10-15 ml of Hanks/RPMI and resuspend cells Take a small sample & perform a viable cell count. NOTE: DILUTE the cell sample 1:10 with Hanks/RPMI first Centrifuge again for 10 minutes at º C, but at a slower speed ( x g) to remove excess platelets.

5 Page: 5 of While centrifuging, calculate the total No. of cells harvested At this point, required cells can be washed into experimental media Do 2x 10mL washes, spin at 1000rpm for 5 mins at RT. Then set up cells for use in experiments to be run immediately (either plates or flasks), and placed into 37ºC incubator for 1-2 hrs prior to drug treatment (for Annexin, cytotoxic assays and PP2A activity assays), or immediately harvested as untreated cell pellets for westerns The remaining cells can be frozen down for later use Calculate the volume of freezing mix to resuspend remaining cells in. Note: aim for 5 x 10^7 cells per ml Remove supernatant, and resuspend cells in freezing mix Transfer cell/freezing mix suspension to cryovials aim for ~ half tubes containing 5x10^5 cells (1ml cells/freezing mix suspension per tube), and half tubes containing 2.5x10^5 cells (ie: 500 µl cell/freezing mix suspension & 500 µl plain freezing mix per tube). Place into Mr Frosty, and put in -80 freezer. Transfer to liquid nitrogen storage after hrs Note: any remaining cells from the cell count sample, can be used to make cytospins for May-Grunwald staining/ihc/if. 8.4 Reviving frozen cells: Thaw cells in a 37 degree C water bath until just a little bit of ice remains. Then transfer immediately to a tube containing mls of thaw media at 4 º C by adding dropwise WORK QUICKLY Centrifuge at 1000rpm for 5 mins at 4 º C Wash again in ml thaw media at 4 º C After washes, resuspend in 10 ml thaw media at 4 º C. Leave cells at RT to equilibrate (for approximately 30 minutes), before placing cells at 37 º C for another minutes NOTE: if using cells to make cell pellets for westerns, etc (eg: PP2A subunits with NO FTY treatment), or doing flow cytometry (no drug treatments), leave the cells at 4 º C (ie: do not warm to 37 º C). If doing survival assays or experiments on FTY treated cells, they will need to be warmed to 37 º C & incubated.

6 Page: 6 of For experiments, once cells at 37 º, wash cells x2 into 10 ml of experimental media 0.5% FBS IMDM + pen/strep, and leave at 37 º C for 1-2 hrs before using for experiments Perform a viable cell count using trypan blue, & calculate required volume of media to achieve desired cell concentration for plating out/harvest cells for experiments The majority of cell lysis will occur as the cells warm from 4-37 ºC. NOTE: If cell viability is poor after thawing (<30%) you can remove the dead cells and debris by layering ficoll under the cells (30ml cells with 20 ml Ficoll in a falcon tube), and spinning at 2000 rpm for 30 mins with the BRAKE OFF. Remove supernatant & collect the mononuclear cell layer, wash 3 times in experimental media (IMDM, 0.5% FBS + pen/strep). Resuspend in 10 ml experimental media, perform viable cell count, add required volume of experimental media to achieve desired cell concentration & plate out/harvest cells for experiments. 9. Maintenance: Shutdown: Waste Disposal: Refer to GLP102 Working with human, murine and GM cell lines. 12. Check List: References: Review Date: Change History: Issue Number: 1st Issue Date Issued:

7 Page: 7 of Issue Number: Date Issued: Reason for Change: