I am looking to separate out erythrocytes from whole horse blood for use in culturing flukes. I have come across a few protocols but most are looking to acquire plasma or leukocytes. Firstly all I want are the erythrocytes (red blood cells), will I require a gradient (Ficoll or Percoll)? What G should I centrifuge at to provide the best separation? Will I need to perform a two-step centrifugation following the removal of plasma/Leukocytes to ensure the other products are free? If anyone has experience with packed red blood cells what is the step to perform this?
Many thanks in advance for your contributions and help.
Hi Adam
A first possible step would be to centrifuge at relatively low revs (looks like about 500g) and then pipette off the plasma and buffy cell layer. That will remove most of the leucocytes. See https://www.researchgate.net/post/What_speed_should_I_centrifuge_whole_blood_at_to_separate_and_measure_for_plasma_free_hemoglobin for various suggestions for another project
Method
1.centrifugation and separation
You an follow two step centrifugation (First slow than fast )
If still you are not getting pure RBCs than do saline wash to remove Plasma and leukocytes.
best wishes
Dharmesh Chandra Sharma
Would you start with 500g? What is the upper limit for RBC centrifugation?
1 what is the quantity of your whole blood .
1. what is maximum speed of your centrifuge
you can adjust the speed Slow means ( you get the separate zones of RBC at bottom than Buffy coat { leukocytes } than platelet rich plasma and on the top platelet poor plasma ) high speed means you get only two zones i.e. Cellular component and plasma .
we are doing that in blood bank with human blood. quantity is 450 ml or so .
slow speed means 1000 to 1500 rpm. high speed means 3000 rpm .
Dharmesh Chandra Sharma We're looking to centrifuge a maximum of 50ml of whole blood in 50ml falcon tubes in a centrifuge with 25,830 x g/15,200 rpm maximum speed. I will be able to run the centrifugation as you've recommended. Many thanks for the help!
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