• AIS MicroCellector anti-CD8 T-25 flasks (AIS, 650139E)

• Dulbecco’s Phosphate Buffered Saline/Calcium Magnesium Free (D-PBS/CMF)

• Ethylenediaminetetraacetic Acid (EDTA)

• Human Gamma Globulins, Cohn fraction II, III (Sigma G4386)

• cIMDM (10% FBS, Pen/Strep)

Solutions:

• D-PBSE/CMF: D-PBS/CMF containing 1mM EDTA

• D-PBSE/CMF/0.5% Ig: dilute human gamma globulins to 0.5% in D-PBS/CMF, heat at 56°C for 30 minutes. Cool to 2-10°C, add EDTA to 1mM final concentration, filter sterilize over 0.2 µm filter.

Protocol:

1. Add 10 ml D-PBSE/CMF to AIS MicroCELLector T-25. Swirl.

2. Incubate for 1 hour at room temperature.

3. Shake vigorously for 30 seconds and aspirate D-PBSE/CMF.

4. Add 10 ml D-PBSE/CMF and shake flask again. Remove D-PBSE/CMF. Repeat 2 additional times.

5. Resuspend PBMC in 4 ml P-DBSE/CMF/0.5% Ig per 20 x 10⁶ cells.

6. Incubate for at least 15 minutes at room temperature.

7. Just before adding the PBMC to flask completely aspirate remaining D-PBSE/CMF from flask.

8. Slowly add 20 x 10⁶ PBMC in 4 ml per flask.

9. Incubate for 1 hour at room temperature (binding of CD8⁺ cells).

10. Gently rock flask from side to side.

11. Collect non-adherent CD8^- cells. Gently wash flask with 4 ml of D-PBSE/CMF. Repeat 3-5 times. Combine all CD8^- cells.

12. Spin at 1500 rpm for 10 minutes, resuspend pellet in cIMDM, count and cryopreserve.

13. Add 5-10 ml cIMDM to the flask.

14. Incubate for 48-72 hours at 37 °C in 5% CO2.

15. Collect CD8⁺ cells. Wash flask with 5-10 ml cIMDM. Combine all CD8⁺ cells.

16. Spin at 1500 rpm for 10 minutes, resuspend pellet in cIMDM, count and cryopreserve

For specifics on appropriate handling and waste procedures please see the online chemical SOPs or our waste and spill notebook located in room 352.

• Ficoll-Paque Plus, 6x 500 ml, Amersham #17144003, $295*

• Dulbecco’s Phosphate Buffered Saline (DPBS), 500ml, BioWhittaker #17-512F, $5.87

• ACK lysing buffer, 100 ml, BioWhittaker #10-548E, $10.80

• Sterile plastic transfer pipette, 500/case, VWR #14670-114, $43

• 50 ml conical tubes, 300/case, ISC #C-3317-6, $98

• Optional: Tri Sodium Citrate Dihydrate (TNC), 38 g/l*

To order buffycoats from American Red Cross, Pacific Northwest Regional Blood Services in Portland: call Hospital Services at 503-284-7008 (option 1). Need Mullins Lab PO number, FedEx account number, budget number and UWATTS code.

Protocol:

1. Transfer blood from bag to sterile 250 ml bottle.

2. Dilute with DPBS to 150 ml total volume.

3. Fill six 50 ml tubes with 12.5 ml Ficoll-Paque Plus each.

4. Gently pipette 25 ml of the diluted cell suspension on top of each 12.5 ml of Ficoll-Paque Plus.

5. Spin 20 minutes at 2000 rpm. No brake!

6. Transfer the white blood cell ring fraction to a new 50 ml tube using a sterile Pasteur pipette. Combine 2 rings into one 50 ml tube.

7. Adjust the volume to 50 ml per tube using PBS.

8. Spin 10 minutes at 1700 rpm.

9. Discard the supernatant.

10. Resuspend each pellet in 2 ml ACK lysing buffer to lyse remaining erythrocytes.

11. Incubate for no more than 2 minutes at room temperature.

12. Adjust the volume to 50 ml using PBS.

13. Spin 10 minutes at 1200 rpm.

14. Resuspend the pellet in 1x PBS. At this point, cells are in two or three tubes; combine cell pellets in 10 ml PBS, rinse tubes with another 10 ml and add to the rest.

15. Count the cells.

Notes:

Ficoll-Paque Plus and PBS should be at room temperature. 10% TNC (v/v) in PBS can be used if erythrocytes are a problem (e.g., with macaque blood). This protocol can also be used to get rid of dead cells: use 2.5 ml Ficoll in 15 ml conical tube, layer 10 ml cell suspension on top, follow protocol from step 5.

TNC is added to increase red blood cell aggregation and decrease monocyte adherence to the tubes. However, the US buffycoats reacted differently and the TNC was not sufficient to remove the red cells, so we included a step with LCK lysing buffer to remove them. Since monocyte yield was not a problem we decided to discontinue the use of TNC for the US buffycoats.

Provided by Mycoplasma Plus PCR Primer Set (Stratagene, #302008, $299):

• Rehydration buffer, store at RT
• PCR primers, rehydrate with 200 µl rehydration buffer, store at -20°C
• Internal control, rehydrate with 100 µl rehydration buffer, store at -20°C
• Positive control, rehydrate with 100 µl rehydration buffer, store at -20°C
• StrataClean Resin, store at 4°C

To be supplied by user:

• 0.6 ml tubes or 1.5 ml screw cap tubes
• PCR reagents (i.e. dNTP, Biolase Taq, MgCL₂,10x reaction buffer)
• UV-irradiated ddH₂O
• 2% agarose gel (3 g agarose, 3ml 50x TAE, 147 ml H₂O, 75 µl (0.5 µg/µl) Ethidium Bromide)
• loading buffer

**Protocol
**

A) Template Preparation

1. Centrifuge all supernatant samples to pellet any cell debris as this can inhibit the PCR.
2. Transfer 100 µl cell supernatant to a 0.6 ml tube (screw cap tubes for viral supernatants). Close tube tightly to prevent opening during heating step.
3. Boil (or heat to 95°C) supernatant for 5 minutes. Spin tube briefly (2-5 seconds) in a microcentrifuge.
4. Resuspend the StrataClean Resin by vortexing for 30 seconds until no pellet remains. Add 10 µl of resin to the extract. Mix the resin and the extract by gently flicking the tube. Briefly centrifuge for 5-10 seconds to pellet the resin.
5. Remove the supernatant to a fresh tube and dilute (optional) 1:10 with UV-irradiated water Per PCR, 5 µl of diluted template is required. Avoid aspirating the resin into the aliquot. Store the template at 4°C until use.

B) Mycoplasma PCR

The amount for one reaction for Mycoplasma PCR is 50 µl. Prepare a premix of reagents for multiple reactions. The amount for the premix reagents is 45 µl per reaction. The amount of each reagent for premix is as follows:

Water 32.1 µl

10x RB 5.0 µl

MgCl₂ (25 mM) 3.0 µl

DNTP (20 mM) 0.5 µl

Primers (25 µM) 2.0 µl

Internal control 2.0 µl

Biolase (5 U/µl) 0.4 µl

45 µl/reaction

1. Calculate volume of premix needed for all samples, one positive control, one negative control (water) and one extra reaction.
2. Thaw PCR reagents from freezer (except Biolase Taq). Make premix in one tube.
3. Add Biolase Taq into premix tube, mix and aliquote into PCR tubes.
4. Add 5 µl test sample, positive control or negative control into the tubes.
5. If not using thermal cycler with heated lid, add 2 drops of mineral oil.
6. Keep tubes on ice until ready for cycling.
7. Start PCR cycling. The following PCR program yields optimal amplification of the 874bp product:

1. Store samples at 4°C until ready to run agarose gel.

2. Make 150 ml of 2% agarose gel.

3. Prepare samples for loading: 2 µl 5x loading buffer, 3 µl H₂O, 5 µl PCR products (or 2 µl 1kb, φx174 or HindIII marker).

4. Load the samples and run the gel for 1 - 1.5 hours at 100 volts.

5. Visualize banding pattern: positive control = 874bp, internal control = 1000bp.

   For specifics on appropriate handling and waste procedures please see the online chemical SOPs or our waste and spill notebook located in room 352.

• Appropriate culture media (i.e. cRPMI for T cell lines, cDMEM for attaching cells) warmed in 37° C waterbath.

• cRPMI = RPMI + 10% FBS+ L-Glut + P/S.

• cDMEM = DMEM + 10%FBS+ L-Glut + P/S + HEPES

• Sterile PBS warmed in 37° C waterbath.

• Antibiotics/proteins/growth factors as needed

Protocol:

Check all cell lines under the microscope.

For Attaching Cells

1. Draw off all supernatant with a pipette. Take care not to disturb the cell layer.

2. Wash once, gently, with PBS to remove any debris.

3. Add just enough trypsin to cover the cell layer (~2.5mL in T75, ~0.5mL in T25).

4. Incubate for 2 minutes MAX at RT. Shake and tap occasionally to verify that the cells are releasing.

5. Add 10mL of cDMEM and mix the cells thoroughly by pipetting up and down until there are few clumps.

6. Draw all of the cell suspension into the pipette, and then leave 1mL in the flask.

7. Discard the rest or save for an experiment.

8. Add 25mL of fresh media.

9. Write new passage number on flask. Take care not to leave the flask upright, as the cells will begin to reattach with the new media.

For Suspension Cells

1. With a pipette, mix the cells thoroughly by pipetting up and down, making sure to rinse the side of the flask.

2. Draw all of the cell suspension into the pipette, and then leave 1mL in the flask. Discard the rest or save for an experiment.

3. Add 10mL of fresh media.

4. Write new passage number on flask.

Appropriate culture media

GHOST cells

• DMEM + 10% FBS + L-Glut + P/S + HEPES

• 500 mg/ml G418

• 100 mg/ml Hygromycin B (reduce to 50mg/ml if cells appear too sensitive)

• Coreceptor expressing cells only (i.e. not parental): 1mg/ml Puromycin

U87 cells

• DMEM + 10% FBS + L-Glut + P/S + HEPES

• 300 mg/ml G418

• Coreceptor expressing cells only (i.e. not parental): 1mg/ml Puromycin

HELA cells

• DMEM + 10% FBS + L-Glut + P/S + HEPES

• Coreceptor expressing cells only (i.e. not parental): 0.4mg/ml Puromycin

For specifics on appropriate handling and waste procedures please see the online chemical SOPs or our waste and spill notebook located in room 352.

• Appropriate cold culture media (i.e. cRPMI for T cell lines, cIMDM for PBMC, cDMEM for attaching cells).

• Sterile DMSO

• Cells to be frozen.

• Freezing vial (1.8 ml, Costar 2028)

• Mr. Frosty controlled freezing container.

Protocol:

1. Make up 20% DMSO/media mixture. Keep cold.

2. Spin down cells at appropriate speed (T cell lines 5 minutes at 1200rpm, PBMC 7 minutes at 1700rpm etc.).

3. Resuspend cell pellet in cold culture media, using half the volume needed. Keep on ice.

4. Prepare freezing vials with name, date and cell number.

5. Drop-by-drop add cold 20%DMSO/media to full volume.

6. Aliquot into freezing vials and cap.

7. Transfer vials into Mr. Frosty.

8. Take Mr. Frosty to -70° C freezer.

9. After overnight freeze, transfer vials to liquid N² tank for long term storage.

10. Make appropriate changes to liquid N² storage log.

For specifics on appropriate handling and waste procedures please see the online chemical SOPs or our waste and spill notebook located in room 352.

• Appropriate cold culture media (i.e. cRPMI for T cell lines, cIMDM for PBMC, cDMEM for attaching cells).

• 50 ml conical tubes (thaw max 3 vials /tube).

• Sterile plastic and glass Pasteur pipettes.

• 10 and 25 ml pipettes.

• Vial(s) with cells to be thawed.

Protocol

1. Perform procedure swiftly, minimizing the time cells are in warm 10% DMSO media.

2. Thaw cells by putting vial in 37° waterbath, until just a small ice-clump remains.

3. Using sterile Pasteur pipette, transfer the cells from vial into labeled 50 ml conical.

4. Slowly add the cold media and shake tube gently while adding in the media. Dilute 10% DMSO media at least 1 in 20.

5. Centrifuge cells at appropriate speed (T cell lines 5 minutes at 1200rpm, PBMC 7 minutes at 1700rpm etc.).

6. Aspirate media carefully, using sterile glass Pasteur pipette.

7. Resuspend cell pellet in 5 ml of appropriate media.

8. Use sample for counting.

9. Make appropriate changes to liquid N² storage log.

• EndoFree Plasmid Maxi Kit, 10 preps, Qiagen #12362, $218

• Effectene Transfection Kit, Qiagen #301425, $205 (contains 1 ml Effectene Transfection Reagent, 0.8 ml Enhancer, 2x 15 ml Buffer EC, sufficient for 8 transfections in 100 mm dishes, store at 2-8°C)

• 100 mm dishes, 200/case, VWR #25382-166, $99.81

• 1.5 ml Eppendorf tubes, 1000/pack, Sarstedt #72690, $16.50

• 15 ml conical tubes, 500/case, ISC #C-3317-1, $70.00

• Appropriate growth medium (e.g., cRPMI for T cell lines)

Protocol:

1. Prepare plasmids for transfection using Endofree Plasmid Kit to avoid toxicity problems.

2. Day before transfection: split cells, need 0.5-2.0 x 10⁷ cells per 100 mm dish.

3. Day of transfection: harvest cells by centrifugation, remove the medium, and wash cells once with PBS in a 15 ml tube.

4. Seed 0.5-2.0 x 10⁷ cells per 100 mm dish in 7 ml growth medium containing serum and antibiotics.

5. Dilute 4 µg DNA (dissolved in TE buffer) with the DNA-condensation bufferEC to a total volume of 600 µl.

6. Add 32 µl Enhancer and vortex for 1 second.

7. Incubate 2-5 minutes at RT, centrifuge briefly to remove drops from top of tube.

8. Add 120 µl Effectene Transfection Reagent. Mix by pipetting up and down 5 times or vortexing for 10 seconds.

9. Incubate 5-10 minutes at RT to allow transfection-complex formation.

10. Add transfection complexes to tube containing 3 ml growth medium. Mix by pipetting up and down twice then immediately add mixture drop-wise onto the cells in the 100 mm dish.

11. Gently swirl the dish to ensure uniform distribution of transfection complexes.

12. Incubate cells with transfection complexes under normal growth conditions for an appropriate time for expression of the transfected gene.

13. Assay cells to confirm gene expression (e.g. X-gal staining, FACS).

For specifics on appropriate handling and waste procedures please see the online chemical SOPs or our waste and spill notebook located in room 352.

For specifics on appropriate handling and waste procedures please see the online chemical SOPs or our waste and spill notebook located in room 352.

2.   Seal off vacuum when done.

3.   Empty vacuum trap flask when full.

4.   Fill bleach bottle when empty

5.   Clean hemocytometer after use.

6.   No liquid in biohazard bag.

7.   Put glass pipettes fully in sharps container.

8.   Fold new pipette boxes when full.

9.   Discard full pipette boxes.

10.   Keep opened tip boxes sterile