2.3. PBMC Isolation, Culture, and RNA Extraction

Detailed Protocol: PBMC Isolation, Culture, and RNA Extraction

I. PBMC Isolation

1. Collect peripheral blood samples in EDTA tubes. 
2. Gently invert the tubes several times to ensure proper mixing. 
3. Dilute blood 1:2 with HEPES-containing buffer. 
   • For each 9 mL blood tube, bring the final volume to 25 mL in a 50 mL Falcon tube using buffer. 
4. Add 12.5 mL Ficoll to a new 50 mL Falcon tube. Carefully layer the 25 mL diluted blood over the Ficoll. 
5. Centrifuge at 2300 rpm for 20 min, with acceleration and brake set at medium level (level 5). 
6. Carefully collect the PBMC layer and transfer to a clean 50 mL tube. Fill up to 50 mL with buffer. 
7. Centrifuge at 1400 rpm for 10 min, with no brake and no acceleration. 
8. Discard the supernatant and resuspend the cell pellet in 10 mL culture medium. 
9. Mix 20 µL cell suspension with 20 µL trypan blue and count viable cells using a microscope/hemocytometer. 

II. PBMC Culture and LPS Stimulation

1. Prepare the LPS working solution. 
2. Add 25 µL LPS to each of the 6 stimulation wells. 
3. Seed 1 × 10⁶ PBMCs into each well (6 control wells + 6 LPS wells) and adjust the final volume to 1 mL per well with culture medium. 
4. Clearly label both the plate lid and the plate bottom. 
5. Incubate cells overnight (~24 h) at 37°C, 5% CO₂. 

III. Cell Harvesting and Lysis

1. After 24 h incubation, remove the plate from the incubator. 
2. Resuspend cells in one control well and one LPS well, then perform a cell count (20 µL cells + 20 µL trypan blue) for quality control. 
3. Centrifuge the plate and transfer supernatants into labeled Eppendorf tubes if storage is required. 
4. Prepare fresh lysis buffer: 

• 1000 µL lysis buffer 
• 20 µL β-mercaptoethanol 

1. Add approximately 100 µL lysis buffer mixture per well (total ~700 µL per pooled sample). 
2. Thoroughly scrape the bottom of each well using a pipette tip to detach all cells. 
3. Transfer pooled lysates into sterile Eppendorf tubes: 

• Control wells → one tube 
• LPS wells → one tube 

IV. RNA Extraction

Kit: GeneJET RNA Purification Kit (Thermo Scientific, Ref. K0731)

1. Add 450 µL of 96% ethanol to each tube containing 700 µL lysate. Mix thoroughly by pipetting. 

• Blank control: 700 µL lysis buffer + 450 µL ethanol 

1. Transfer 700 µL of the mixture to the RNA spin column. 
2. Centrifuge 1 min at 12,000 rpm. 
3. Discard flow-through and repeat until the full sample has passed through the column. 
4. Transfer the column to a new collection tube. 
5. Add 700 µL Wash Buffer 1. 
6. Centrifuge 1 min at 12,000 rpm and discard flow-through. 
7. Add 600 µL Wash Buffer 2. 
8. Centrifuge 1 min at 12,000 rpm and discard flow-through. 
9. Add 250 µL Wash Buffer 2. 
10. Centrifuge 2 min at 12,000 rpm and discard flow-through. 
11. Perform an additional dry spin for 1 min at maximum speed. 
12. Transfer the column to a clean RNase-free collection tube. 
13. Add 50 µL RNase-free water directly onto the membrane. 
14. Centrifuge 1 min at 12,000 rpm to elute RNA. 
15. Store isolated RNA at −80°C until downstream analysis. 

Supplementary Solutions

V. HEPES-Containing Buffer

Materials

• HBSS 10× medium 
• Measuring cylinder 
• Distilled/demineralized water 
• HEPES powder 
• pH meter 
• Sterile bottles with filter 

Method

1. Add 200 mL HBSS 10× to a measuring cylinder. 
2. Add 1800 mL distilled water (final volume = 2 L). 
3. Add 12 g HEPES. 
4. Adjust pH to 7.4 using NaOH or HCl. 
5. Sterile filter and aliquot into 500 mL sterile bottles. 

VI. Culture Medium Preparation

Materials

• RPMI 1640 medium 
• Fetal bovine serum (FBS) 
• Antibiotics (AB/AM) 
• Sterile filter 

Method

1. Transfer RPMI 1640 medium into sterile filter unit. 
2. Add 50 mL FBS. 
3. Add 5 mL antibiotics. 
4. Filter sterilize and return to storage bottle. 

VII. LPS Stock Solution

Materials

• LPS powder 
• Distilled water 
• 50 mL Falcon tube 
• 1.5 mL Eppendorf tubes 
• Stirring magnet 

Method

1. Gently open the LPS vial. 
2. Prepare stock at 1 mg/mL in distilled water. 
   • Example: for 10 mg LPS, add 10 mL water. 
3. Mix thoroughly and allow to dissolve for at least 30 min. 
4. Confirm complete dissolution. 
5. Aliquot 500 µL into 1.5 mL Eppendorf tubes. 
6. Store frozen until use. 

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