Cell sample collection

Cell culture SAMPLES

  • Remove the medium.
  • Rinse with PBS or cold saline (+4°C).
  • Scrape the cells in the presence of PBS or cold (+4°C) saline serum.
  • Transfer them in a conical tube.
  • Centrifuge at 1500 rpm/5 minutes.
  • Gently aspirate as much supernatant as possible in order to obtain the driest possible pellet.
  • Place the tube in ice
  • Freeze the tube as soon as possible. (if possible transfer to – 80°C as quickly as possible).

 

 

Blood cell samples

Draw two 5 mL EDTA tubes or vacutainers.

Red blood cells and plasma

  • Use one 5 mL EDTA tube
  • Centrifuge at + 4 °C (if possible) at approx. 3000 rpm for 10 minutes.
  • Separate the plasma: separate into three aliquots of 500 µL each.
  • Wash the pellets of the globules with saline + 4 °C (0.9% NaCl), volume to volume.
  • Shake by inversion
  • Centrifuge at approx. 3000 rpm, + 4 °C if possible, 5 minutes.
  • Remove supernatants by aspirating the interface of white blood cells and platelets (e.g. using soft plastic pipettes).
  • Repeat the washing operation once.
  • Divide into 3 fractions, > 100 microliters each
  • Freeze plasma and cell fractions at – 20 °C as quickly as possible (if possible transfer quickly to – 80°C).

White blood cells, lymphocytes

  • Use the second 5 mL EDTA tube
  • Dilute a volume of blood with the same volume of NaCl 0.9%, (1V/1V)
  • Pour diluted blood over 1V of lymphoprep®. Pay attention to the deposit. It must be performed as gently as possible, inverting the drops, drop by drop for the first mL, on the wall of the tube, as close as possible to lymphoprep®. There should be no mixing between diluted blood and lymphoprep®. This is an important prerequisite for the success of the subsequent separation.
  • Centrifuge 4000 rpm 20min at 20°C (avoid cold centrifuge)
  • Remove diluted plasma
  • Pipette (white) lymphocytes layer.
  • Transfer to an eppendorf tube
  • Wash the cells with at least 1 mL saline + 4 °C (0.9% NaCl).
  • Shake by inversion
  • Centrifuge at approx. 3000 rpm, + 4 °C if possible, 5 minutes.
  • Remove as much supernatant as possible with a pipette, in order to have as little residual volume as possible.
  • Freeze as quickly as possible at -20°C (if possible quickly transfer to – 80°C).

 

 

 

 

 

Tissue specimen collection

 

Biopsies should be frozen as soon as possible.

  • Punch biopsy
  • Place it as soon as possible in a freezer tube (NUNC tube) for liquid nitrogen.
  • Dispose of this tube in liquid nitrogen (thermos tanks).
  • Transfer the tube to the freezer (if possible transfer quickly to – 80°C).

 

 

 

Specimen transfer

 

  • Transport specimen in dry ice or any other means that ensures a temperature below – 10°C.