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  Protocol: Lipid Nanoparticle (LNP) Preparation 1. Preparation of Lipid Solution In a 50 mL centrifuge tube, mix the following lipid stock solutions in ethanol: Component Stock Conc. Volume Ionizable lipid 5 mM 300 µL Helper lipid 5 mM 60 µL Cholesterol 5 mM 231 µL DMG‑PEG2000 1 mM 45 µL Total Volume   636 µL 2. Preparation of mRNA Solution Dissolve 40 µg of mRNA in 1.9 mL of 20 mM Malic or Citric Acid Buffer (pH 3.0). 3. LNP Assembly Quickly add the mRNA solution into the lipid solution while vortexing for 10 s. Incubate the mixture at room temperature for 10 min. 4. Dilution and Buffer Exchange Add 30 mL of TBS buffer to the mixture to form the LNP solution. 5. LNP Concentration (1-2 hours) - Transfer 15 mL of the LNP solution to an Amicon® Ultra‑15 mL 100 kDa...

  MTT Assay Protocol 1. Prepare MTT Solution (5 mg/mL in PBS) ·        - Filter sterilize the solution using a 0.22 μm filter. ·        - Aliquot and store at –20°C (stable for at least 6 months). 2 . Discard Culture Media ·        - Adherent cells: Carefully aspirate the media. ·        - Suspension cells: Centrifuge the plate at 1,000 × g, 4°C, 5 min, then carefully aspirate media. 3 . Add Reagents ·        - Add 50 µL serum-free media to each well. ·        - Add 50 µL MTT solution (5 mg/mL) to each well. ·        - If culture medium contains serum or phenol red, set up background control wells with 50 μL MTT solution + 50 μL media (no cells). ·          Incubate at 37°C for 3 hours. ·     ...

  Protocol: T Cell Isolation and Activation from Mouse Spleen and Lymph Nodes 1. Tissue Harvesting and Cell Suspension Preparation • Isolate all lymph nodes (LNs) and spleens from 7–8 mice. • Place tissues in isolation buffer (500 mL PBS + 2.5 g BSA + 2 mL 0.5 M EDTA) in a 6 cm dish on ice. • Complete step 2-6 within 1 hour to maintain cell viability. 2. Tissue Dissociation • Grind spleens and LNs on a 70 µm strainer fitted into a 50 mL tube using the plunger of a 1 mL syringe. • Place 4–5 spleens on one strainer, remaining spleens and all LNs on the second. • Flush with 5 mL isolation buffer multiple times (≥4–5), alternating with grinding. • Also flush isolation buffer from dish and rinse dish with 5 mL isolation buffer into the strainers. • Final volume per tube ~30 mL. 3. Red Blood Cell Lysis • Centrifuge tubes at 420 g for 8 min at 4–10°C. • Discard supernatant and resuspend pellet in 2 mL ACK lysis buffer, incubate at RT for 5 min. • Add 10 mL isolati...

  Protocol: In Vitro Transcription (IVT) for mRNA Synthesis 1. Template Preparation (Linearization and PCR Amplification) a. Linearization of Plasmid - Digest 5–10 µg of pUC57AG-GOI (e.g., pUC57AG_Luc) using XbaI at 37 °C. - Run the digested product on a 1% agarose gel (60 mL TAE buffer + 0.6 g agarose + 6 µL GelRed). - Load: 5 µL DNA + 1 µL DNA loading dye; Run at 120 V for 30 min. - Purify the linearized product using a commercial PCR Purification Kit. - Quantify the DNA using Nanodrop, adjust to 1  ng/µL, and name it as L_pUC57AG_GOI. b. PCR Amplification (to add polyA tail) Use a KOD hot-start kit for PCR: Component Volume (µL) 10× KOD buffer 5 25 mM MgSO₄ 3 2 mM dNTP mix 5 5′ Primer (10 µM) 1.5 3′ Primer (10 µM) 1.5 Template DNA (12.5 ng) 12.5 KOD Polymerase 1 Nuclease-f...