NK Cell Culture Medium (cRPMI for Murine NK Cells)

Dillon Corvino

Complete NK culture medium (cRPMI) for murine NK cells, including RPMI 1640 supplemented with FCS, antibiotics, HEPES, sodium pyruvate, NEAA, and beta-mercaptoethanol. IL-15 is added fresh at the time of culture and is not included in the stored base medium.
Published

September 18, 2026

Protocol ID: BUF-NK-MED Version: v1.1 Author: Dillon Corvino Date: September 18, 2026

Purpose

This medium is used for in vitro culture and proliferation assays of murine NK cells. It is based on complete RPMI 1640 supplemented with nutrients essential for lymphocyte viability and proliferation. IL-15 is added fresh immediately before plating cells and is not included in the stock medium.

Linked protocols

  • MUS-SPL-NK-PROLIF
  • GEN-LYMPH-NUC

Working buffer composition

Component Final concentration Notes
RPMI 1640 — Base medium
Fetal calf serum (FCS) 10% (v/v) Heat-inactivated
Penicillin/Streptomycin 1% (v/v) 100x stock -> 1x final
HEPES 10 mM (~1% v/v) Buffering at 37 °C
Sodium pyruvate (NaPy) 1 mM (1% v/v) Energy substrate
Non-essential amino acids (NEAA) 1% (v/v) Supports lymphocyte metabolism
beta-mercaptoethanol (beta-ME) 50 uM (e.g. 0.05 mM) Reduces oxidative stress
Note

IL-15 is not included in this stock medium. It must be added to a final concentration of 100 IU/mL immediately before culturing NK cells.

Preparation

A. Preparation of stock (if applicable)

Not applicable — prepared directly as the working buffer from commercial stock reagents. Stock reagents required:

Reagent Stock concentration Notes
RPMI 1640 — Sterile-filtered
FCS, heat-inactivated — 10% final
Pen/Strep 100x 1% final
HEPES buffer 1 M 10 mM final (~1% v/v)
Sodium pyruvate (NaPy) 100 mM 1 mM final (1% v/v)
NEAA 100x 1% final
beta-mercaptoethanol 50 mM or 14.3 M See dilution below

B. Preparation of working buffer

Preparation of 500 mL NK medium (cRPMI base):

  1. In a sterile bottle, combine:
    • 450 mL RPMI 1640
    • 50 mL FCS (10% final)
  2. Add supplements:
    • 5 mL Pen/Strep (100x stock -> 1% final)
    • 5 mL HEPES (1 M stock -> 10 mM final)
    • 5 mL Sodium pyruvate (100 mM stock -> 1 mM final)
    • 5 mL NEAA (100x stock -> 1% final)
  3. Add beta-mercaptoethanol:
    • Final concentration: 50 uM
    • If using 50 mM stock: add 0.5 mL
    • If using 14.3 M stock: prepare a working dilution before adding
  4. Mix gently by inversion; do not vortex.
  5. Label with:
    • “NK medium (cRPMI base)”
    • Date of preparation
    • Expiry (4-5 weeks at 4 °C)
Warning

beta-mercaptoethanol is hazardous. Handle it in a fume hood and avoid skin contact. Do not open concentrated beta-ME bottles inside a biosafety cabinet.

Usage

  • Warm medium to 37 °C before adding cells.
  • Add IL-15 fresh immediately prior to plating NK cells:
    • Final concentration 100 IU/mL.
  • Do not store IL-15 in the base medium.

Storage and stability

  • Store at 4 °C, protected from light.
  • Use within 4-5 weeks of preparation.
  • Do not freeze.

Reagent details

Reagent Supplier Cat. # Notes
RPMI 1640 Various — Base medium
FCS Various — Heat-inactivated
Pen/Strep Various — 100x working stock
HEPES Various — 1 M stock
Sodium pyruvate Various — 100 mM stock
NEAA Various — 100x stock
beta-mercaptoethanol Sigma/etc — Handle with caution
IL-15 (added fresh) PeproTech/etc — Not included in base medium

Safety (brief)

  • Handle beta-mercaptoethanol and all supplements using gloves and eye protection.
  • beta-ME requires fume hood handling due to volatility.
  • Prepare medium under aseptic conditions inside a biosafety cabinet.
  • Dispose of expired or contaminated medium as biohazardous waste.

Version history

Version Date Author Changes
v1.0 2025-11-21 Dillon Corvino Initial Quarto buffer document for NK medium
v1.1 2026-09-18 Dillon Corvino Migrated to shared summary:/helper architecture (buffer/protocol redesign).