Homo sapiens (lymphocytes)

(Cryopreservation of Human Lymphocytes)

Intro/Description:

This protocol is from a study which sought to determine effects of mutation in the human MUTYH gene on DNA damage binding and repair.  In their study, they collected and cryopreserved lymphocytes.  We report their protocol.

Materials:

•  Blood collection tube (50 ml or larger)

•  RPMI-1640 medium

•  Tri-sodium citrate

•  Mercapthoethanol

•  CaCl2

•  Conical glass flask

•  Sterile glass beads

•  Gyrotory shaker (New Brunswick Scientific)

•  Lymphoprep™ solution (Axis-Shield Diagnostics)

•  Fetal calf serum (FCS)

•  Dimethylsulfoxide (DMSO)

•  Liquid nitrogen 

Protocol:

1.  Collect 25ml peripheral blood into a tube containing 25 ml of RPMI-1640/0.6% tri-sodium citrate/0.04mM mercapthoethanol.

2.  Maintain at room temperature during shipment to the laboratory (up to 72 h).

3.  Add 600 µl of 1M CaCl2.

4.  Defibrinate the blood in a conical glass flask containing sterile beads by shaking at 250 r.p.m. for 15 min on a Gyrotory shaker.

5.  Isolate lymphocytes from the defribinated blood using Lymphoprep™ solution according to the manufacturer’s instructions.

6.  Count the isolated cells and re-suspend in 1 ml of cryopreservation solution consisting of 90% FCS / 10% DMSO.

7.  Freeze the samples at -80OC.

8.  Store in liquid nitrogen.

9.  Thaw the frozen lymphocyte suspension at 37°C.

10.  Wash in 10 ml RPMI-1640/20% FCS. Your cells are now ready for downstream use.

References:

Anthony R.Parker, Oliver M.Sieber, Chanjuan Shi, Li Hua, Masashi Takao, Ian P. Tomlinson and James R. Eshleman. Cells with pathogenic biallelic mutations in the human MUTYH gene are defective in DNA damage binding and repair. Carcinogenesis. 2005;26(11):2010-2018

Added by: Carlton Hoyt on September 26, 2012

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