Purification, freezing, and thawing of 3D7 P. falciparum-infected red blood cells and antibody-dependent cellular cytotoxicity (ADCC ) assay

Jagannathan Lab, Stanford University

Savannah Nicole Lewis (snlewis@stanford.edu)

I: Purification and freezing of 3D7 P. falciparum-infected red blood cells

-Materials:

• Purification:
  • Incomplete and complete RPMI: at room temperature
  • Pf cultures at late trophozoite to early schizont stage
    • Transfer 10mL Petri dish culture to 15mL conical tube, centrifuge (1500rpm, 5 minutes), remove supernatant, resuspend in 6mL iRPMI
  • Magnet columns with plungers: LS or LD (preferred) columns
    • Note: one column can only filter 20mL of Pf culture
  • Trypan Blue dye
• Freezing:
  • Glycerolyte
  • FBS

-iRBC purification:

• Block LD columns with 3mL cRPMI and let completely pass through into 50mL conical tubes
• Add 6mL Pf culture resuspension to columns, avoid creating any bubbles
• When finished dripping, wash column three times with 3mL iRPMI
• Remove column from magnetized stand and place under a clean 15mL conical tube
• Add 5mL iRPMI to top of column and use plunger to elute iRBCs
• Resuspend elution well and centrifuge (1500rpm, 5 minutes)

-Freezing purified iRBCs:

• Aspirate supernatant and use a pipette tip/adjust volume to determine the volume (V, in uL) of packed iRBCs
  • Avoid losing cells by cleaning the pipette tip in a separate container of iRPMI
• Bring volume up to 5mL, resuspend pelleted iRBCs, and centrifuge (1500rpm, 5 minutes) while counting:
  • Remove 10uL from 5mL suspension, add to 10uL Trypan Blue
  • Mix and count either manually (hemocytometer) or automatically (Countess Cell Counter)

• Aspirate as much supernatant as possible from 15mL conical iRBC tube
• Add 25uL FBS to pellet, then add 1/3V glycerolyte and rest for 5 minutes
• Add remaining 2/3V glycerolyte to pellet, mix well, then aliquot desired amount into pre-labeled cryovials
  • Note: expect to lose ~half of frozen amount when thawing

• Put cryovials in a Mr. Frosty in the -80 freezer overnight, then transfer to liquid nitrogen freezer the following day

II: Antibody-dependent cellular cytotoxicity (ADCC) assay

-Materials:

• Thawing frozen iRBCs:
  • MTS (Malaria thawing solution): 3.5% NaCl
  • 50:50 MTS/PBS: 1.8% NaCl
  • 1X PBS
  • Frozen Pf iRBCs
  • R10 media
• Opsonizing iRBCs:
  • RPMI media
  • Thawed and counted iRBCs: concentration of 5M per 100uL
  • Opsonins: plasma, antibody
• Co-incubation of PBMCs and RBCs:
  • R10 media
  • GolgiPlug (brefeldin): 0.12uL per well
  • GolgiStop (monensin): 0.12uL per well
  • Optional: fluorophore-conjugated degranulation antibody

-Thawing frozen Pf iRBCs:

• Quickly bring frozen iRBCs to room temperature by thawing in your hands
• Add 20uL MTS slowly and dropwise while flicking the cryovial, then rest for 5 minutes
• Add 1mL MTS slowly and dropwise while flicking cryovial
• Transfer iRBCs to 15mL conical tube and then centrifuge (1500rpm, 5 minutes)\
• Aspirate supernatant and resuspend pelleted RBCs by flicking
• Add 1mL 50:50 MTS slowly and dropwise while flicking tube, then centrifuge (1500rpm, 5 minutes)
• Aspirate supernatant and resuspend pelleted RBCs by flicking
• Add 1mL PBS slowly and dropwise while flicking tube, then centrifuge (1500rpm, 5 minutes)
• Aspirate supernatant and resuspend pelleted RBCs by flicking
• Raise 15mL conical tube volume to 5mL with R10
• Remove 10uL resuspended cells, add to 10uL Trypan Blue, and count cells either manually (hemocytometer) or automatically (Countess Cell Counter)

-Opsonizing iRBCs:

• Pre-label 15mL conical tubes according to opsonizing conditions
• Split iRBCs into opsonizing conditions (adjust accordingly based on volume and number of cells):
  • Plasma (10%): 90uL RPMI + 10uL plasma
  • Antibody (1%): 99uL RPMI + 1uL antibody
• Centrifuge (300g, 10 minutes)
• Incubate (37°C, 5% CO₂ ) for 1 hour, flicking tubes to mix every 15 minutes (4 times total)
• After 1 hour, raise total 15mL conical volume to 500uL in RPMI and centrifuge (300g, 10 minutes)
• Aspirate supernatant, resuspend in 500uL RPMI, and centrifuge (300g, 10 minutes)
• Resuspend in appropriate volume needed for desired effector to target ratio (ex: 1:2, etc.)

-Co-incubation of PBMCs and opsonized RBCs:

• Take plated PBMCs out of incubator and centrifuge (1500rpm, 5 minutes)
• Aspirate supernatant and resuspend cells in 100uL R10
  • Optional: add degranulation antibody at this step
• Add 100uL RBCs at concentration for desired effector to target ratio
• Add ER inhibitors to all wells and then centrifuge plate (100g, 3 minutes)
• Incubate for 5 hours (37°C, 5% CO₂ )