2x Freezing Medium (R0 + 10% FCS + 20% DMSO)

Dillon Corvino

Preparation of 2x freezing medium for cryopreservation of PBMCs and other lymphocytes. Mixed 1:1 with cell suspension to yield standard 10% DMSO freezing medium.
Published

September 18, 2026

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

Purpose

This buffer is a 2x concentrated freezing medium used for the cryopreservation of PBMCs and other lymphocytes. When mixed 1:1 with a cell suspension in R10 or R0, it yields a standard final freezing medium containing 10% DMSO and 5% FCS (or 10% FCS if starting from R10), suitable for controlled-rate freezing.

Linked protocols

  • HUM-BLD-PBMC-ISO
  • HUM-NK92-CULTURE
  • HUM-KHYG1-CULTURE
  • HUM-K562-CULTURE

Working buffer composition

Component Final concentration (2x) Purpose
RPMI 1640 (R0) — Base medium
Fetal calf serum 10% (v/v) Protects cells during freezing
DMSO (cell-grade) 20% (v/v) Cryoprotectant

When mixed 1:1 with cells in R10 or R0, the final freezing medium contains:

  • 10% DMSO
  • 5–10% FCS (depending on suspension medium)
  • RPMI base medium

Preparation

A. Preparation of stock (if applicable)

Not applicable — prepared directly as the working buffer from off-the-shelf stock reagents (see table below).

Reagent Stock concentration Notes
RPMI 1640 — Sterile-filtered
DMSO, cell-grade 100% Use fresh or freshly opened
Fetal calf serum — Heat-inactivated recommended

B. Preparation of working buffer

Preparation of 10 mL 2x freezing medium:

  1. In a sterile 15 mL tube, add:
    • 7 mL R0 medium (RPMI without serum)
    • 1 mL heat-inactivated FCS (10% v/v)
    • 2 mL DMSO (20% v/v)
  2. Mix gently by inversion; do not vortex.
  3. Keep on ice until use.
Warning

DMSO is toxic at room temperature. Keep the 2x freezing medium on ice, and mix with cells immediately after preparation.

Usage

  1. Adjust PBMCs or lymphocytes to 20 x10^6 cells/mL in R10 or R0.
  2. Add an equal volume of this 2x freezing medium.
  3. Mix gently by pipetting.
  4. Dispense 1 mL per cryovial (final 10% DMSO).
  5. Freeze using a controlled-rate freezing container (e.g. Mr Frosty) at −70/−80 °C.
  6. Transfer to liquid nitrogen after ≥12 hours.

Storage and stability

  • Use fresh on the day of preparation.
  • Keep on ice during use.
  • Do not store after the experiment; discard any remaining buffer.
  • DMSO-containing mixtures degrade rapidly and should never be refrozen.

Reagent details

Reagent Supplier Cat. # Notes
RPMI 1640 [TBD] [TBD] Base medium
Fetal calf serum [TBD] [TBD] Heat-inactivated
DMSO, cell-grade [TBD] [TBD] Highly hygroscopic; handle aseptically

Safety (brief)

  • Handle DMSO with gloves; it enhances dermal absorption of other chemicals.
  • Prepare freezing medium in a biosafety cabinet using aseptic technique.
  • Dispose of DMSO-containing waste according to institutional chemical waste protocols.
  • Avoid eye/skin contact and inhalation.

Version history

Version Date Author Changes
v1.0 2025-11-21 Dillon Corvino Initial Quarto buffer document for 2x freezing medium.
v1.1 2026-09-18 Dillon Corvino Migrated to shared summary:/helper architecture (buffer/protocol redesign); added Storage/Safety/Reagent-details sections.