Feb 25, 2020

Public workspacePreparation of leukocytes by differential lysis of erythrocytes

DOI
dx.doi.org/10.17504/protocols.io.bcv2iw8e
  • Mesut Karataş1,
  • Şenol Doğan2,
  • Emrulla Spahiu3,
  • Adna Ašić1,
  • Larisa Bešić1,
  • Yusuf Turan1
  • 1International Burch University;
  • 2Universität Leipzig;
  • 3Middle East Technical University
Emrulla Spahiu
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DOI: dx.doi.org/10.17504/protocols.io.bcv2iw8e
Protocol Citation: Mesut Karataş, Şenol Doğan, Emrulla Spahiu, Adna Ašić, Larisa Bešić, Yusuf Turan 2020. Preparation of leukocytes by differential lysis of erythrocytes. protocols.io https://dx.doi.org/10.17504/protocols.io.bcv2iw8e
Manuscript citation:
Enzyme Kinetics and Inhibition Parameters of Human Leukocyte Glucosylceramidase
Mesut Karataş, Şenol Doğan, Emrulla Spahiu, Adna Ašić, Larisa Bešić, Yusuf Turan
bioRxiv 2020.02.23.961599; doi: https://doi.org/10.1101/2020.02.23.961599
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 23, 2020
Last Modified: February 25, 2020
Protocol Integer ID: 33434
Keywords: human, leukocyte,
Abstract
Leukocytes are isolated by centrifugation after specific lysis of erythrocytes
Materials
REAGENTS
1. Lysis Buffer (155 mmol/L ammonium chloride; 10 mmol/L sodium bicarbonate; 0.1 mmol/L EDTA).

Dissolve 8.3 g ammonium chloride, 0.84 g sodium bicarbonate and 29.3 mg EDTA in about 900 mL reagent-grade water. Titrate to pH 7.4 with HCl, then make the volume to 1 litre. Store at 4oC.

2. Isotonic Saline (0.9%, w/v)

Dissolve 9 g NaCl in 1 litre reagent-grade water. Store at 4 degrees Celsius.
Centrifuge Amount10 mL EDTA blood to pellet all cells Centrifigation1500 rpm, 4°C, 00:10:00
10m
Remove plasma into a clean container and freeze.
Restore original blood volume with 0.9% saline and transfer the blood suspension into a Amount50 mL conical centrifuge tube.

Add Amount40 mL cold lysis buffer
Stand TemperatureOn ice , mixing occasionally, until erythrocytes are lysed (the red cell suspension remains red in colour, but becomes transparent). This should take only about Duration00:05:00 to Duration00:10:00
10m
Centrifuge Centrifigation1500 rpm, 4°C, 00:05:00
5m
Discard supernatant and resuspend leucocyte pellet in Amount5 mL cold lysis buffer.
Stand TemperatureOn ice for Duration00:10:00
10m
Dilute cell suspension to Amount50 mL with cold 0.9% saline. Mix and centifuge Centrifigation1500 rpm, 4°C, 00:05:00
5m
Discard supernatant, then resuspend leucocyte pellet in Amount10 mL 0.9% saline. Care should be taken to obtain an even cell suspension without being too vigorous and causing cell disruption.
Divide the cell suspension into two equal aliquots, into two Amount10 mL conical centrifuge tubes.
Centrifuge Centrifigation1500 rpm, 4°C, 00:05:00
5m
Remove all supernatant and dry walls of centrifuge tube with a tissue.
Store leucocyte pellets and plasma at Temperature-20 °C (or Temperature-80 °C )