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now: Sat Feb 24 14:35:59 2018 ... mod: Tue Jan 3 04:46:40 2017
Preparation of Leukocytes by Differential Lysis of Erythrocytes
Leukocytes are isolated by centrifugation after specific lysis of erythrocytes.
- After the procedure has been initiated the blood/leukocytes must be kept on ice at all times. The exception is the centrifugation steps, which ideally will be at 4C, but which will still work fine at room temperature if a refrigerated centrifuge is not available. The leukocytes become much less stable and prone to lysis if allowed to warm up, but if kept on ice are reasonably robust and easy to manipulate.
- The entire procedure, start-to-finish, should take place in one 50 ml conical tube per sample. This will help minimize losses in transfer between tubes. If done carefully, the recovery of total leukocytes can be better than 50%.
- This protocol is designed for whole-blood volumes of 10 ml or less. The procedure can be scaled up suitably as necessary.
- It is often useful to perform a QuickLeukocyteAnalysis on the whole blood before beginning the leukocyte isolation to be able to calculate the percent yield of leukocytes upon isolation.
- Make sure anti-coagulated whole blood is well-suspended by gentle inversion, pour the resuspended blood into a 50 ml conical tube. If the volume of blood is less than 10 ml, add ice-cold 0.9% saline to a total volume of 10 ml and swirl gently to mix.
- blood needs to be anti-coagulated. EDTA for this purpose is slightly preferable to heparin
- Add 40 mL ice-cold lysis buffer and mix completely by gentle inversion.
- Stand on ice, mixing occasionally, until erythrocytes are lysed
- the red cell suspension will remain red in colour, but become both transparent and a darker shade of red. this phase change when it happens is very obvious -- if you are unsure, leave the cell suspension on ice a little longer.
- the complete lysis should take only about 5-10 minutes. the lysis process does not happen gradually but in a rather sudden phase-transition after an appropriate time on ice.
- Centrifuge at 1,500 g for 5 minutes.
- Discard supernatant by careful vacuum aspiration.
- Resuspend the leukocyte pellet thoroughly in 1 mL ice-cold lysis buffer by repeated pipetting with a P1000 tip on a pipetman. After the cells have been homogeneously resuspended, add an additional 4 ml ice-cold lysis buffer, swirl gently to mix, and stand on ice for 10 minutes.
- it is important to resuspend the cell pellet in a small volume first to effectively be able to make a homogenous cell suspension. any clumpiness will reduce final yield
- Dilute cell suspension to 50 mL with ice-cold 0.9% saline. Mix and centrifuge 1,500 g for 5 minutes.
- Discard the supernatant by gentle vacuum aspiration. Resuspend the leukocyte pellet thoroughly in 1 mL ice-cold 0.9% saline by repeated pipetting with a P1000 tip on a pipetman until the cell suspension is completely homogeneous, then add an additional 9 ml ice-cold 0.9% saline and swirl gently to mix
- Count the yield of cells.
- a 40:1 dilution counted by flow cytometry is generally reasonable. There should be relatively clear separation between the leukocyte populations and platelets/erythrocyte ghosts on a SSC vs FSC plot. The leukocytes have larger values for both SSC and FSC. Be sure that the concentration of cells is not too high for the cytometer to count accurately - make a larger dilution if this is the case.
- even better: stain the leukocytes with AcridineOrange at a final concentration of 2 μg/ml. Cells containing DNA will fluoresce green in the presence of AO. The green fluorescence provides even better distinction between debris/ghosts and leukocytes
- Centrifuge at 200 g for 5 minutes to concentrate the leukocytes.
- note the much more gentle spin for this final concentration step
- Remove all supernatant by careful vacuum aspiration.
- if there are beads of liquid remaining the sides of the tube, these can also be aspirated
- Resuspend the leukocytes to an appropriate concentration with ice-cold 0.9% saline, or alternatively store leukocyte pellets at -20 degrees Celsius (or -80).
- if making HighMwGenomicDna, resuspend the leukocyte pellet to a final concentration of 3x107/ml in ice-cold 0.9% saline and transfer to an eppendorf tube on ice. Be certain to account for the volume of the pellet and any residual liquid in the resuspension step to avoid having a cell suspension that is lower density and higher volume that what you had anticipated.
Both solutions should be made fresh, placed in glass bottles in ice-water baths and occasionally swirled until their temperature has fully equilibrated to that of the ice-water bath. Keep these solutions on ice for the duration of the procedure.
- The use of glass rather than plastic will help speed the temperature equilibration.
Lysis Buffer (155 mmol/L ammonium chloride; 10 mmol/L sodium bicarbonate; 1 mmol/L EDTA)
- Dissolve 8.3 g ammonium chloride, 0.84 g sodium bicarbonate and 2.0 ml 500 mM EDTA (pH 8.0) to a final volume of 1 liter in water.
- prepare this solution fresh before every use. apparently dissolved CO2 in water will convert NH4Cl to (NH4)2CO3 and reduce the effectiveness of the erythrocyte lysis
Isotonic Saline (0.9%, w/v)
- Dissolve 9 g NaCl in 1 litre reagent-grade water.
National Referral Laboratory for Lysosomal, peroxisomal and Related Genetic Disorders, Department of Chemical Pathology, Women's & Children's Hospital, Adelaide, Australia
- also possible is isolation of leukocytes by differential sedimentation in either dextran or ficoll gradients