SAMPLES PREPARATION

Blood Cells and Plasma Preparation

Red Cells and Plasma Preparation

– Sample container : 1 x 5 ml lithium heparinate tube.

– Spin 3000 rpm 10 minutes 4 °C

– Transfer 2 x 1 ml aliquots plasma in separated glass tubes.

– Wash the Red Cell pellets with normal saline solution (NaCl 0.9%) at 4 °C vol/vol.

– Agitate by inverting several times to suspend the cells.

– Spin 3000 rpm 5 minutes 4 °C

– Discard the supernatant (plasma and buffy coat).

– Wash the Red Cell pellets with normal saline solution (NaCl 0.9%) at 4 °C vol/vol.

– Dispatch in 3 aliquots, > 200 µL each.

– Freeze the plasma and Red Cell aliquots at -20°C as quickly as possible. Transfer immediately at -80°C, if possible.

sample preparation

WBCs Preparation

– Collect blood into a 5 ml lithium heparinate tube.

– Pipette 4 ml blood in a 10 ml glass tube.

– Dilute the blood by addition of an equal volume of 0.9% NaCl.

– Carefully (http://youtu.be/Cl4sTwKFe9k) layer 1 volume of the diluted blood over 1 volume Lymphoprep™ (or Polymorphprep™, or Percoll solution with adequate density…) in a 12–15 mm centrifuge tube. Avoid mixing of blood and separation fluid. Cap the tube to prevent the formation of aerosols.

– Centrifuge at 600 x g for 20 minutes at room temperature (approximately 20˚C)

– Discard the supernatant (diluted plasma).

– After centrifugation the lymphocytes (or Polynuclear cells, monocytes,…) form a distinct white band at the sample/medium interface. The cells are best removed from the interface using a Pasteur pipette

– Transfer the lymphocytes (or Polynuclear cells, monocytes,…) in an eppendorf tube.

– Dilute the harvested fraction with at least 1ml 0.9% NaCl at + 4°C to reduce the density of the solution.

– Agitate by inverting several times to suspend the cells.

– Pellet the cells by centrifugation for 5 minutes at 3000 rpm, + 4°C if possible.

– Discard the supernatant.

– Freeze the Cells at -20°C as quickly as possible. Transfer immediately at -80°C, if possible.

Cultured cell preparation

– Cells must be washed two times in cold PBS

– After washing, scrapped the cells using either rubber policeman or adequate device then transferred the cells in a 15 mL Falcon polypropylene tube

– Pellet the cells by centrifugation at 3000 rpm, +4°C if possible

– Discard supernatant

– Freeze the cells at -20°C as quickly as possible. Transfer immediately at -80°C, if possible.

Cells from Biopsies or Tissues

– Freeze as soon as possible with no particular preparation

– Please contact us in order to define the necessary quantities for your study

Liquids from diverse origins

– Freeze as soon as possible with no particular preparation

– Please contact us in order to define the necessary quantities for your study

SAMPLES EXPEDITION

Frozen samples must be sent to Metabiolab Lab in dry ice.