Hi Julie, We use 0.5 M EDTA (Gibco) at a ratio of 1:10, i.e. 4 ul EDTA for 40 ul whole blood. It is very important to rapidly and vigorously mix the blood with the EDTA solution when working in these very small volumes to prevent clotting. I have found that it is quite acceptable to leave the mouse blood as whole blood for up to two days. Within this time there are minimal/undetectable changes in expression of CD4, CD8, TcR-beta, and CD45R/B220. I routinely analyze whole blood shipped overnight with good quality results. -Jim Weaver ************************************************* * * * James L. Weaver Ph.D. * * Division of Applied Pharmacology Research * * Office of Testing & Research * * CDER MOD-1, FDA * * 8301 Muirkirk Rd, Laurel MD 20708 * * * * Phone: 301-827-8237 * * Fax: 301-594-3037 * * Email:WEAVER@CDER.FDA.GOV * * * * This email is my personal opinion and is NOT * * in any way official U.S. FDA Policy * ************************************************* * -----Original Message----- * From: Oughton, Julie [mailto:julie.oughton@orst.edu] * Sent: Thursday, March 21, 2002 12:42 PM * To: Cytometry Mailing List * Subject: RE: method of blood collection (Thanks and summary) * * * * Thanks for posting the summary to the entire group. Even * though I'm more * interested in mouse blood, the information is quite * pertinent and very * interesting. * * However, I would like to entertain a follow-up question. * * I am involved in a project with the SMILE program (Science and Math * Investigative Learning Experiences) where we will be introducing flow * cytometry to a group of high school girls. We will examine * the blood for * various leukocyte phenotypes and compare the results with * blood smears and * WBC. * * Typically, I collect and phenotype mouse blood all in one * day. Due to the * lack of time, we will need to collect blood samples the day * before the SMILE * project. Hence, we need a good anti-coagulant which will * allow us to hold * samples overnight. * * I've searched the email archives for the most appropriate * anticoagulant for * blood storage. From what I could glean in the archives, * sodium heparin may * be our best choice. Any comments or suggestions? * * Thanks in advance, * Julie Oughton * Oregon State University * * * * -----Original Message----- * From: MIGUEL-ANGEL PERALES [mailto:mperales@OPAL.TUFTS.EDU] * Sent: Tuesday, March 19, 2002 12:29 PM * To: cyto-inbox * Subject: Re: method of blood collection (Thanks and summary) * * * * * * Question: Does the method of blood collection (green tops, CPT, * leukapheresis) influence results of T cell assays such * as Elispot? * * Conclusions: ACD may be better for straight phenotype, * but heparin is * probably better for T cells used in functional assays. * Based on our * experience, this may also be true for T cells used for Tetramer * assays. * * Responses: * Russ Wick: Apheresis instruments always collects blood in ACD. * Heparin tubes are used for various immunological * assays. Examples * * include mixed lymphocyte culter and mitogen response. * My suspecion is * that ACD has some effect of lymphocyte response. Only a guess... * * Jeffrey Harris: the sodium heparin green top tubes can activate * lymphocytes- that's why many labs use lavender/purple * tops (using EDTA * as anticoagulant) or yellow top ACD (acid * citrate/dextrose - citrate * is also an anticoagulant) tubes for culture/stimulation * methods and * even phenotyping when you are worried about a labile * marker - I must * say however, that for robust assays I don't think the * heparin is a * huge effert, because we use them for the CFCs in the * core lab and our * "non-stimulated" controls for cytokine secretion are * quite negative, * but there is some small background activation by * markers like CD69; * similarly we sort samples based on CD62L, which is also * quite labile * and sensitive to stimulation/temperature/etc. but we * try to use the * blood freshly and move quickly to separate the * lymphocytes by Ficoll * and keep them cold on ice to avoid surface marker * changes - whereas * the core and clinical labs use only EDTa-containing tubes for * phenotyping for this reason. * * Keith Bahjat: It is true that sodium heparin * vacutainers may contain * varying amounts of endotoxin, leading to unwanted * cellular activation, * especially of APCs. I remember hearing a while back that BD was * supposed to be working on certified endotoxin-free * vacutainers, but I * haven't seen anything yet. But in my experience, this kind of * contamination is minimal to nonexistent. When blood is * collected and * then leukopheresed, CPDA is the anticoagulant used. * CPDA, like EDTA * and ACD, is a calcium chelator. Lymphocytes collected in * calcium-chelating agents are extremely difficult to * activate (we all * know what role calcium mobilization plays in cellular * activation, so I * won't elaborate). Several rounds of washing with a * calcium containing * medium can restore some of this potential, but they * still fall far * short of cells which have never been exposed to * calcium-chelators. I * wouldn't recommend use of calcium-chelators for any * activation-dependant experiments, no matter how many * washing steps * were involved. * * *
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