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Collecting Blood Samples Page 5 of 8 (Version 030626) MicroFlow BASIC Kit (Mouse) Method: Collect mouse peripheral blood using a method approved by the Institutional Animal Care and Use Committee (IACUC). The tail vein is a common site for obtaining peripheral blood from a rat. The subsequent fixation procedure and micronucleus analysis is also effective for blood obtained from the orbital sinus or by cardiac puncture. Volume: Collect only 60–120 µl of blood (whether taking blood from the tail vein, orbital sinus, or heart) from each animal. It is important to obtain no more than 120 µl of blood since too much blood may compromise the flow cytometric analysis. It is also important that no debris be introduced into the sample. Anticoagulant: Use only Solution B as the anticoagulant; do not substitute a different anticoagulant. Collect drops of blood from the tail vein directly into tubes of Solution B. If a capillary tube or syringe is used to draw blood, pre-wet it with Solution B. Have extra tubes containing Solution B ready in case of a spill. Warning: Some capillary tubes include a clot activator that will cause aggregation and make the samples unanalyzable. 1. Tubes containing 0.35 ml Solution B (anticoagulant) should be stored at 4°C until use. Approximately one hour before collecting blood, move these tubes to room temperature and maintain at room temperature throughout the blood collection procedure. 2. For tail vein bleeding, place a mouse or group of mice (up to 5 animals) under a heat lamp (100 watt bulb placed 6 to 8 inches above the animals) for 5–10 minutes to dilate the tail veins. 3. Shake the microcentrifuge tube immediately before bleeding each animal to coat the inside of the tube with Solution B. 4. Make an incision in the tail vein using a sterile blade. 5. Place the corresponding labeled tube of Solution B under the incision, and collect approximately three drops of blood (~120 µl). [Again, too much blood can be as great of a problem as not having enough. The volume of Solution B is appropriate for approximately three drops of blood. Varying from this amount may lead to samples which cannot be analyzed.] To prevent clotting, hold the tube so that the blood drops directly into Solution B and does not contact the side of the tube. It is important to make the incision such that the blood is free flowing, and falls directly into Solution B in a minimal amount of time (< 10 seconds). Apply moderate pressure to the wound with a laboratory tissue to stop the bleeding. 6. Cap the tube and invert the tube several times to mix the blood with Solution B. The blood/Solution B mixture can be stored at room temperature for up to 6 hours before fixing. 7. Repeat steps 3–6 for the remaining mice. Fixing Conditions It is extremely important that the tubes containing Solution A and fixed blood remain ultracold (–75 to –85 °C) and do not come in contact with vapors from dry ice. CO2 vapor causes carbonation and cellular aggregation. For this same reason, fixative should not be stored in a freezer containing dry ice, and fixation should not occur on dry ice. To avoid this problem, Solution A should be taken directly from the freezer. If you are unable to fix blood samples DIRECTLY from the –75 to –85 °C freezer as described in the Fixing Procedure, it is imperative that you do not fix the samples off dry ice under any circumstances without first calling Litron at (585) 442-0930 or sending an email to info@litronlabs.com. Litron will provide you with a detailed alternative fixing protocol. Without this alternative protocol, blood samples CANNOT be fixed appropriately and will be compromised, and flow cytometric analysis CANNOT be performed. Litron Laboratories is not responsible for samples prepared using a procedure other than the one described in this manual or supplied by Litron as an alternative.
Fixing Procedure Solution A must be kept in an ultracold (–75 to –85 °C) freezer. To prevent Solution A from excessive warming, perform the following steps very quickly (less than one minute) and work near the ultracold freezer. It is preferable to perform the fixing procedure from a chest freezer because they maintain temperature better than upright freezers. Page 6 of 8 (Version 030626) MicroFlow BASIC Kit (Mouse) Initially, it may be helpful for two individuals to perform this procedure, one removing blood from the collection tubes and the other removing the fixative tubes from the freezer. In this case, two people would be able to fix two blood samples in the same time it would normally require to fix one blood sample. 1. Immediately prior to fixing, invert the tube containing the blood sample to ensure a homogeneous suspension. 2. Using a pipettor, retrieve 180 µl of the blood sample. 3. Remove the corresponding labeled 15 ml tube containing 2 ml of Solution A from the freezer and uncap. 4. Holding the tube upright, with the label and graduation marks turned away so that the inside of the tube is clearly visible, position the pipette tip approximately 1 cm above the surface of the ultracold Solution A. Making sure that the pipet tip does not touch the side of the tube or the surface of Solution A, forcefully dispense the blood sample into Solution A. 5. Recap the tube. While holding the top of the tube in one hand, sharply strike the bottom of the tube with your other hand approximately 5 times to discourage aggregate formation. Check that the cap is on securely, and immediately transfer the tube to the –75 to –85 °C freezer for storage. Blood fixed in Solution A must be maintained in a –75 to –85 °C freezer until preparation for flow cytometric analysis. 6. Repeat steps 1–5 for the remaining samples. There should be enough volume in each tube to fix every sample twice. The duplicate (backup) samples are important in the event that flow cytometric analysis problems arise. 7. If the freezer temperature begins to warm up significantly (i.e., raises by 5 °C), stop processing samples. Wait until the freezer temperature returns to the required range before completing sample fixation. Remember that blood collected into Solution B can be stored at room temperature for up to 4 hours before fixing. Again, due to their ability to hold temperature better, chest freezers are recommended. 8. Store all of the samples at –75 to –85 °C for at least 24 hours before shipping. Samples stored in Solution A in a –75 to –85 °C freezer are stable for at least 1 year, as long as temperature has been maintained. 9. Ship samples to Litron for flow cytometric analysis as described below. Shipping Samples to Litron for Analysis 1. Please ship only one set of samples to Litron for analysis. Leave the duplicate samples in storage at –75 to –85 °C until analysis is complete. 2. Immediately before shipping, ensure the tubes are capped tightly to prevent diffusion of CO 2 into the samples during shipping. Do this quickly so that the samples do not warm up. Make sure all tubes are in racks and upright before sealing the kitsupplied heavy-duty plastic bags. A video of the fixing procedure (QuickTime format) is available on our website: www.litronlabs.com. Please use the insulated shipping container received with the kit for shipment of samples to Litron. If necessary, a second box can be obtained from Litron. 3. Complete and sign the Protocol Acceptance Form. Seal this document inside the small clear plastic bag. Sample analysis cannot begin until this document is received. The document is also available on our website at www.litronlabs.com. 4. Move the dry ice and shipping container next to the freezer where the samples are stored. [It is important to utilize the shipping container received with the kit. If you need to use another shipping container, be sure that it has adequate insulating capacity and sufficient room for dry ice.] The volume of dry ice should be twice the volume taken up by the samples. Using less dry ice than recommended can compromise your samples during shipment.
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