Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Basic Lab Protocols and Techniques
Register Search Today's Posts Mark Forums Read


Washing Red blood cells

Washing Red blood cells - Basic Lab Protocols and Techniques

Washing Red blood cells -


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 08-02-2010, 01:00 AM
Pipette Filler
Points: 32, Level: 1 Points: 32, Level: 1 Points: 32, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2010
Posts: 4
Thanks: 0
Thanked 0 Times in 0 Posts
Default Washing Red blood cells



Any tips on washing red blood cells and removing the plasma and buffy coat?

I'm collecting blood in EDTA tubes, centrifuging them at 1500 x G for 15 minutes and washing with saline. Having a hard time getting all of the buffy coat. I think I ended up lysing the red blood cells as well. Had a slight conical shaped red film developing at the top of the sample against the side of the test tube (was this lysed cells?). Thanks, any tips would be appreciated.
Reply With Quote
  #2  
Old 08-03-2010, 06:20 PM
luisillo's Avatar
M.D/Ph.D
Points: 4,061, Level: 42 Points: 4,061, Level: 42 Points: 4,061, Level: 42
Activity: 25% Activity: 25% Activity: 25%
 
Join Date: Jul 2010
Location: Mexico
Posts: 353
Thanks: 19
Thanked 98 Times in 90 Posts
Default Re: Washing Red blood cells

I don't know if I got it right: you want to keep red cells and discard white ones and plasma? If this is right why don't you try Dextran sedimentation:

1. Collect blood into a tube containing sodium or potassium EDTA as anticoagulant.
2. Mix 10 parts of EDTA-blood with 1 part of 8% (w/v) Dextran 500 in 0.9% (w/v) NaCl.
3. After 15-40 min at room temperature remove the plasma layer, containing the leucocytes. The time taken for the erythrocytes to settle out depends on the individual blood sample, the initial blood column height and the ambient temperature.

This protocol will also helps if you want the opposite, I mean, if you want white cells instead of red ones.

Hope it helps
Reply With Quote
The Following User Says Thank You to luisillo For This Useful Post:
admin (03-12-2011)
  #3  
Old 08-08-2010, 02:05 AM
Pipette Filler
Points: 32, Level: 1 Points: 32, Level: 1 Points: 32, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2010
Posts: 4
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Washing Red blood cells

Ended up spinning it slower and I think I got a hang of removing the buffy coat, thanks for the tips.
Reply With Quote
  #4  
Old 03-01-2011, 01:07 PM
Zagami's Avatar
Ph.D Doctorate
Points: 4,121, Level: 43 Points: 4,121, Level: 43 Points: 4,121, Level: 43
Activity: 25% Activity: 25% Activity: 25%
 
Join Date: Sep 2009
Location: Tabaka Mission Hospital - Kiisi -Kenya & Italy Sicily
Posts: 160
Thanks: 2
Thanked 28 Times in 24 Posts
Default Re: Washing Red blood cells

Freshly drawn blood have a total specific gravity between 1.052 and 1.064. Considering that blood is constituted by formed elements (erythrocytes, leucocytes and platelets) and liquid medium (plasma), every component a different density show. Red cells have a nominal density about 1.10 g/ml, white cells a density about 1.075, and platelets-plasma a density of about 1.027. Mature human erythrocyte show the highest density, despite has lost nucleus, Golgi apparatus, centrioles, ER and most of its mitochondria, and this because both cytoplasm bulk consist (90-95% of dry weight) of the iron-carrying pigment hemoglobin that is responsible of 80-90 % of high specific gravity (1.092-1.101, measured by means benzyl benzoate/cottonseed oil serial mixture) and, for a small part owed to the aggregation of the erythrocytes among them. Among the sweaters of aggregation can trap leucocytes or platelets, what don't totally allow the obtainment a suspension buffy-coat free. Insofar, is necessary to proceed at several and differential washings-centrifugations, without addition of some gradient medium. As anticoagulant as been used EDTA-K3 and whole blood has been centrifuged for 10 min. at 120 g for obtained platelet-rich plasma (PRP). Discard supernatant and add disodium EDTA buffered saline solution (wash chelating solution [WCS] for minimize erythrocytes aggregation) to reconstitute initial blood volume. Mix accurately and centrifuge at 500 g for 10 min., discard supernatant and add WCS to reconstitute initial blood volume. Mix accurately and centrifuge at 1000 g for 20 min. Drain supernatant and package erythrocytes is buffy-coat free with purity > 98 %.
Reply With Quote
The Following User Says Thank You to Zagami For This Useful Post:
admin (03-12-2011)
  #5  
Old 03-01-2011, 01:57 PM
Zagami's Avatar
Ph.D Doctorate
Points: 4,121, Level: 43 Points: 4,121, Level: 43 Points: 4,121, Level: 43
Activity: 25% Activity: 25% Activity: 25%
 
Join Date: Sep 2009
Location: Tabaka Mission Hospital - Kiisi -Kenya & Italy Sicily
Posts: 160
Thanks: 2
Thanked 28 Times in 24 Posts
Default Re: Washing Red blood cells

my mistake!
change ... to reconstitute per 2 times initial blood volume, instead ... to reconstitute initial blood volume
excuse to me
Reply With Quote
The Following User Says Thank You to Zagami For This Useful Post:
admin (03-12-2011)
Reply

Tags
blood , cells , red , washing


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
mouse hepatocytes isolation amicia Cell Biology and Cell Culture 39 05-11-2012 11:52 AM
Characteristics of cancerous cells aftabac Cell Biology and Cell Culture 2 08-26-2011 11:40 PM
Human Cytome Project - Update 24 Jan. 2005 Peter Van Osta Cell Biology and Cell Culture 1 08-01-2010 02:18 PM
How to isolate adult from circulation? peterish Stem Cell Forum 0 05-19-2008 10:02 PM
Human Cytome Project - Update 6 Jan. 2005 Peter Van Osta Cell Biology and Cell Culture 0 01-06-2005 11:18 AM


All times are GMT. The time now is 08:33 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.17538 seconds with 16 queries