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  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...