PROTOCOL FOR IN VITRO ACTIVATION OF HUMAN BLOOD

Notes: Human blood can contain infectious agents. Blood needs to be manipulated in a biosafety cabinet, not on the bench, by properly trained personnel. Applying the aseptic technique will minimize the possibility of external contamination of cultures, materials and reagents. The footnote lists the vendors and catalog numbers for the consumables used in the experiments, but does not represent the endorsement of a particular source over other comparable products.

MATERIALS

• Culture plate: 96-deep well plate, polypropylene (PPN), sterile, endotoxin-free¹
• Sealable lids for 96-deep well plate²
• Sterile lids for 96-deep well plate³
• 96-well V-bottom plate with lid, polystyrene, sterile, endotoxin-free⁴
• 50 mL and 100 mL reagent reservoirs, sterile, single-packed, endotoxin-free⁵
• 96-well PPN tubes (for supernatant storage)⁶
• ACD Vacutainer tubes⁷
• Tempus reagent (from Tempus tubes)⁸
• Medium (RPMI-GlutaMax⁹)
• Stimuli (diluted to final volume of 50 µL/test)
• Single-channel pipettes
• Multi-channel pipettes (200 or 300 µL, and 1200 µL volume)
• Biosafety cabinet, level 2
• CO2 incubator
• Refrigerators (+4^oC, -20^oC, -80^oC)
• Centrifuge with a cooling-option to +4oC and a rotor with plate adapters (2000 x g required)

PROCEDURE

Collection of human blood

1. Calculate the volume of blood needed for the experiment (0.5mL/well) and number of required ACD Vacutainer tubes.
Notes
1. Record transport time (from needle to bench) of ACD tube.
2. In our hands, 0.5 mL of blood yields 1-2 µg of total RNA. The volume of blood/well can be reduced up to 0.2 mL/well.

Preparation of reagents

1. Design plate layout and prepare experiment documentation.
2. Calculate the amount of stimulus to be added per well to obtain the necessary concentration for a total volume of 1 mL (use template table). Dilute each stimulus in total volume of 50 µL of medium per well.
3. Adjust the volumes of diluted stimuli to the number of wells and add 10-15% pipetting overage.
4. Aliquot stimulus-medium mix to the 96-well V-bottom plate which layout that corresponds to the culture plate; the diluted stimuli can be added to the blood with a multi-channel pipette.
5. Cover the plate and keep at +4oC until the stimulation.

Note: It is recommended to create an Excel spreadsheet with all the stimuli, their stock concentrations, working concentrations and volumes, so calculations can be easily adjusted depending on the number of samples.

Preparation of blood-medium mix and culture

1. Calculate the amount of blood needed for all the wells (0.5 mL/well) and add 10-15% pipetting overage.
2. Transfer the diluted stimuli (50 µL/well) to the culture plate.
3. Transfer the blood to the reagent reservoir and add medium in 1.1 to 1 ratio. Mix gently with the pipette. Avoid splatter.
4. Transfer the blood-medium mix (0.95 mL/well) to the culture plate with a multi-channel pipette.
5. Mix gently with a multichannel pipette. The total volume/well is now 1 mL.
6. Culture the plate for 6h in a 5% CO2 incubator set to 37oC.

Note: If the reagents and consumables are not tested for endotoxin, 10 µg/mL of polymyxin B can be added to wells to prevent unintended TLR4 activation with endotoxin (LPS).

Aliquoting blood for flow cytometry

1. Remove the plate(s) from the incubator and mix the wells gently with a multi-channel pipette.
2. Remove 100 µL of blood for flow cytometry staining.

Supernatant harvest

1. Centrifuge the plate(s) at 2,000 x g for 15 min at +4oC.
2. Carefully remove 400 µL of the supernatant with a multi-channel pipette and transfer 2 x 200 µL aliquots in 96-well PPN tubes.
Notes
1. Plates need to be properly balanced and certified to sustain high g.
2. Whole blood pellet is liquid and easily disturbed. 200 or 300 µL pipettes are recommended for aliquoting the supernatant.
3. The number and volume of aliquots can be modified according to individual requirements, providing the final well volume is not exceeded once the Tempus solution is added to the blood pellet in a 2:1 ratio.
3. Label PPN racks, store the supernatants at -80oC and update the storage location log.

Blood lysis and preservation until RNA extraction

1. Calculate the necessary volume of Tempus reagent needed for the plate (Tempus to blood pellet ratio = 2:1); add 10% pipetting overage.
2. Pour the Tempus solution into the reagent reservoir of appropriate size.
3. Using a multi-channel pipette, add 1 mL of Tempus reagent per well (that contains 0.5 mL of blood pellet). Mix well by pipetting vigorously but carefully up and down at least 5 times. Avoid foaming.

Note: After Tempus reagent is properly mixed in, the color of blood changes from deep red to very dark brown with a reddish hue.

4. Close the plate with the sealable lid; label the plate, update sample log form and store at -20oC until RNA extraction.

Note: If the lid does not seal the plate tightly, place the plate in an additional leak-proof biohazard bag or container.

¹ Greiner Bio-One 2mL deep 96-well PP Masterblock plates, sterile, pyrogen-free. USA Scientific, # 5678-0271
² Greiner Bio-One EVA cover for 96-well 2.0 ml Masterblock. USA Scientific, # 5638-1080
³ Thermo Scientific plate lids, sterile individually wrapped. Thermo Scientific, # 62402-929
⁴ Corning 96 well clear V-bottom tc-treated microplate, individually wrapped, with lid, sterile. Corning #3894
⁵ Costar 50 mL & 100 mL Reagent Reservoirs, individually wrapped, sterile. Corning #4871 & 4873
⁶ Micronic 0.5 mL alphanumeric screw cap tubes V bottom in 1D barcoded, Loborack 96 case, Micronic # MPW42049BCX1
⁷ BD 8.5 mL BD Vacutainer glass whole blood tube, ACD solution A. BD # 364606. ACD solution volume = 1.5 mL
⁸ Applied Biosystems Tempus Blood RNA Tubes. Applied Biosystems # 4342792
⁹ Invitrogen RPMI 1640 (1X), liquid, contains GlutaMAX & Phenol Red, 10 × 500 mL. Invitrogen # 61870-127