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University of Washington School of Medicine, Department of Microbiology     

Monocyte-Derived Macrophages Adherence & Susceptibility

Isolation of Monocyte-Derived Macrophages from Buffy Coat by Plastic Adherence

Reagents

  • PBS: Dulbecco's Phosphate Buffered Saline (BioWhittaker, #17-512F)
  • IMDM: Iscoves Modified Dulbecco's Medium (CellGro, #150116CV)
  • ΔHPS: Heat-inactivated human AB pool serum (Irvine Scientific, #5009)
  • ΔFBS: Heat-inactivated fetal bovine serum (NovaTech, Inc, #15-167-P50)
  • P/S: Penicillin/Streptomycin (CellGro #30002CI)
  • L-glutamine (CellGro #25005CI)
  • Polybrene (Sigma, #H-9268)
  • 0.53mM EDTA (GeneMate, #C-5512-500G)
  • DMSO: Dimethyl Sulfoxide (Sigma, # D-5879, Lot 51K0004)
  • PHA: Phytohaemagglutinin (Remel, Murex HA16/17, #30852801)
  • RNAlater (Ambion, #7024)
  • Primaria T25 tissue culture flasks (VWR Scientific, #15705-068)
  • Primaria T75 tissue culture flasks (VWR Scientific, #15705-070)
  • Primaria 96-well flat bottom tissue culture plates (VWR Scientific, #62406-503)
  • Corning 96-well flat bottom tissue culture plates (Corning Inc, Costar, #3596)
  • Cell scrapers (VWR Scientific, #62407-140)
  • Cryovials (Corning Inc, # 2028)

Protocol

Day 1
  1. Isolate PBMC from Buffy Coat (see Isolation of PBMC using Ficoll gradient)
  2. Centrifuge 10 min at 1100 rpm to pellet cells
  3. Resuspend PBMC in 50mL MDM media (500mL IMDM, 10% ΔHPS, 1% P/S, 1% L-glutamine, sterile filtered)
  4. Save PBMC aliquots for FACS (0.5 million cells each)
  5. Divide remaining PBMC in media so that no more than 200 million cells (ideally ~160 million) go into each flask, add to 75 cm2 Primaria flask, adjust media volume in flask to 20mL
  6. Incubate 2 hours at 37°C in 5% CO2
  7. Check adherence under microscope
  8. Remove non-adherent cells (PBL) to 50 mL conical vial
  9. Save PBL aliquots for FACS (0.5 million cells each)*
  10. Rinse 3x with 10 mL IMDM, being careful not to pipet solution against cell layer
  11. Add 20 mL new MDM media
  12. Incubate overnight at 37°C in 5% CO2
* Do not discard remaining PBL, but process as follows:
  1. Count remaining PBL
  2. Centrifuge for 10 min at 1500 rpm to pellet cells, remove and discard supernatant
  3. Combining pellets from same donor, resuspend cells at 100 million/mL in cold PBMC media (IMDM, 10% ΔFBS, 1% P/S, 1% L-glutamine, 5 µg/ml polybrene)
  4. Add 1 volume PBMC freezing media (IMDM, 20% ΔFBS, 20% DMSO) dropwise while shaking
  5. Pipet 1 mL aliquots (50 million) into cryovials, viably freeze
Day 2

Proceed with one donor at a time to minimize waiting during which time cells die or adhere

  1. Remove and discard non-adherent cells
  2. Rinse gently with 10 mL room temp IMDM (2x if many non-adherent cells)
  3. Rinse gently but quickly with 10 mL room temp PBS
  4. Examine under microscope
  5. Detach cells by adding 10 mL room temp PBS/0.53 mM EDTA, incubate 10 min at 37°C in 5% CO2, knocking cells loose as much as possible
  6. Scrape remaining cells loose
  7. Transfer into 50 mL conical vial
  8. Rinse flask with 5 mL PBS, add to 50 mL conical vial
  9. Centrifuge for 10 min at 1100 rpm to pellet cells
  10. Remove and discard supernatants
  11. Combining pellets from the same donor, resuspend in 20 mL PBS
  12. Count:
  13. From 50 mL conical vial of monocytes, withdraw aliquots for FACS (0.5 million each), RNAlater (5 million), T25 flask for MDM RNA (5 million)
  14. Centrifuge for 10 min at 1100 rpm to pellet cells, remove and discard supernatants
  15. Resuspend cells for RNAlater in 100 µL PBS, transfer to Eppendorf tube, add 5 volumes RNAlater, store overnight at 4°C, then transfer to -20°C
  16. Resuspend cells for T25 flask in 5mL MDM media, transfer to Primaria T25 with filter cap, incubate 5-7 days at 37°C in 5% CO2 until TCID50 is started
  17. Resuspend the remaining monocytes in MDM media (0.5 million cells/mL) and aliquot 100 µL/well into Primaria 96-well plates (50,000 cells/well)
  18. Incubate monocytes 5-7 days at 37°C in 5% CO2 before use in TCID50
Day 5
  1. Thaw 1 vial PBL (~50 million cells) by slowly adding 15-20 ml cold PBMC media
  2. Centrifuge 10 min at 1500 rpm to pellet cells, discard supernatant
  3. Resuspend cell pellet in 15 mL PBMC media, transfer to T75 flask
  4. Add 75 µL PHA (1 µg/mL)
  5. Incubate cells 2-3 days at 37°C in 5% CO2 before use in TCID50
Day 7
  1. Transfer PHA-PBL from T75 into 50 mL conical vial
  2. Centrifuge for 10 min at 1500 rpm to pellet cells
  3. Resuspend cell pellet in warm PBMC media
  4. Count cells and adjust culture volume to get 1 million cells/mL
  5. Add rIL-2 at 20 units/mL
  6. Aliquot 100 µL/well in Nunc 96-well plates (100,000 cells/well)
  7. Start TCID50 (8 virus isolates, 3-fold serial dilution series, 8 dilutions, each dilution tested in triplicate, 25µL per well) on both MDM and PHA-PBL in 96-well plates
  8. Isolate RNA from T25 with 5-7 day old MDM and from monocytes stored in RNAlater using Qiagen RNeasy Kit according to manufacturer's instructions.
Day 8

Proceed with one plate at a time to minimize waiting during which time cells die

  1. Using a multichannel and starting from the highest dilution (i.e. lowest virus concentration), remove and discard all culture supernatants from MDM plates, taking care not to cross contaminate cells
  2. Wash wells 2 times with warm PBS
  3. Add 200 µL MDM media per well
  4. Culture for 14 days at 37°C in 5% CO2
Day 12 (TCID50 Day 5)
  1. Thaw 1 vial PBL (~50 million cells) by slowly adding 15-20 ml cold PBMC media
  2. Centrifuge 10 min at 1500 rpm to pellet cells, discard supernatant
  3. Resuspend cell pellet in 15 mL PBMC media, transfer to T75 flask
  4. Add 75 µL PHA (1 µg/mL)
  5. Incubate cells 2-3 days at 37°C in 5% CO2 before use in TCID50
Day 14 (TCID50 Day 7)

Collect supernatants for p24 ELISA and passage cultures:

  1. MDM: collect 75 µL supernatant, discard remaining supernatant, add 200 µL fresh MDM medium to each well
  2. PHA-PBL: collect 65 µL supernatant, transfer 65 µL supernatant and cells to new 96-well plate containing 135 µL new PHA-PBL (100,000 cells/well)
Day 19 (TCID50 Day 12)
  1. Thaw 1 vial PBL (~50 million cells) by slowly adding 15-20 ml cold PBMC media
  2. Centrifuge 10 min at 1500 rpm to pellet cells, discard supernatant
  3. Resuspend cell pellet in 15 mL PBMC media, transfer to T75 flask
  4. Add 75 µL PHA (1 µg/mL)
  5. Incubate cells 2-3 days at 37°C in 5% CO2 before use in TCID50
Day 21 (TCID50 Day 14)
Collect supernatants for p24 ELISA and passage cultures:
  1. MDM: collect 75 µL supernatant, discard remaining supernatant, add 200 µL fresh MDM medium to each well
  2. PHA-PBL: collect 65 µL supernatant, transfer 65 µL supernatant and cells to new 96-well plate containing 135 µL new PHA-PBL (100,000 cells/well)

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

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jl, 8dec04

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