1. Materials Provided Additional Materials Required First-Time Users Ordering Information and Technical Services...

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1. Materials Provided

Kit Component Quantitya Storage Conditionb

Anticoagulant Solution 10 mL 2 °C to 8 °C

Blood Freezing Solution 30 mL Ambient

Cryovials 50 Ambient

Cryovial Storage Boxes 2 Ambient

a. Sufficient materials are provided to collect 25 rodent blood samples and freeze in duplicate.

b. Please note that although kit components are shipped at ambient temperature, they must be stored at the temperatures indicated above upon receipt.

2. Additional Materials Required

• Blood collection supplies

• Depending on blood collection technique, heparin-coated capillary tubes, 115 µl capacity (e.g., RAM Scientific cat # 06 0005), requires 2 per rodent • K2EDTA Microtainer tubes (e.g., BD cat # 365974)

• Micropipettors (20 µL - 1000 µL) and tips

• 2 °C to 8 °C refrigerator

• Labels compatible with ultracold storage (Litron recommends Cryo-Tags® labels)

• –75 °C to –85 °C freezer

• Dry ice (if shipping samples to Litron for analysis)

3. First-Time Users

We strongly recommend reading the entire instruction manual before performing these procedures.

Please do not deviate from the procedures described in this manual. It is important that these steps are followed exactly using the reagents supplied with this kit in order to achieve reliable results. If you have questions, please contact Litron Laboratories by calling (585) 442-0930, faxing us at (585) 442-0934, or sending an email to [email protected].

4. Ordering Information and Technical Services

Litron Laboratories

3500 Winton Place, Suite 1B Rochester, New York 14623

Telephone: 585-442-0930

Order Toll Free: 877-4-LITRON (877-454-8766)

Fax: 585-442-0934

email: [email protected]

World Wide Web: www.LitronLabs.com

5. License Agreement and Limited Product Warranty

By utilizing this kit, your company is agreeing to be bound by the terms of this License. This License allows the use of the MutaFlow® Rodent Blood Freezing Kit for the collection of 25 samples and for freezing in duplicate.

By accepting these products, you acknowledge that they will be used in accordance with their intended labeling (For in

vitro research use only. Not for human or animal diagnostic or therapeutic use.). Uses other than the labeled intended

use may be a violation of local laws.

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6. Introduction

This instruction manual describes procedures for collecting and freezing rodent blood that may subsequently be analyzed for the frequency of mutant phenotype erythrocytes (RBCs) and mutant phenotype immature erythrocytes (reticulocytes, or RETs) using flow cytometry.

The MutaFlow Pig-a analysis method is based on the endogenous Pig-a gene whose product is essential for the synthesis of glycosylphosphatidylinositol (GPI) anchors. Hematopoietic cells require GPI anchors to attach a host of proteins to their cell surface (for instance, CD24, CD55, and CD59). Importantly, of the genes required to form GPI anchors, only Pig-a is located on the X-chromosome. Mutations in the Pig-a gene can prevent functional anchors from being produced, resulting in cells lacking these proteins on their surface. Thus, cells without these cell surface markers represent a reliable phenotypic marker of Pig-a mutation.

7. Overview of Method

The following steps are performed when preparing blood samples for storage using the MutaFlow Rodent Blood Freezing Kit.

8. Collect Whole Blood Samples

Important Notes:

• Use an IACUC approved method to collect whole blood. As described below, the volume collected is dependent on the method used.

• Choose either the technique described in 8.1 or the technique described in 8.2 Whatever blood collection technique is used, it is essential that blood is free-flowing from an animal whose heart is still beating in order to prevent platelet activation and cellular aggregation.

• Collect free-flowing blood sample. Required volumes are specific to the bleeding technique used, and are indicated below.

8.1. Blood Collection into Heparin-coated Capillary Tubes

1. For blood samples collected into heparin-coated capillary tubes, add 40 µL of kit-provided Anticoagulant Solution to each EDTA tube BEFORE blood is transferred to these tubes. Label each tube with the animal identification number.

2. Collect blood.

a. If blood will be collected by nicking a tail vein with a surgical blade, it is important to warm the animals under a heat lamp for several minutes. Once the blood starts flowing, use heparin-coated capillary tube(s) to collect approximately 200 µL/rodent. We recommend heparin-coated capillary tubes from RAM Scientific, cat # 06 0005.

b. If blood will be obtained via submandibular puncture using a lancet, or via retro-orbital bleed, it is important to collect the free-flowing blood into heparin-coated capillary tube(s) (approximately 200 µL/rodent).

Move blood samples into Freezing Solution and then transfer to an ultracold freezer, see Section 9.

Store frozen samples until you are ready for thawing and Pig-a analyses using MutaFlow Rodent Blood Thawing Kit and MutaFlowPLUS Kit, each sold separately; or until you are ready to ship frozen samples on

dry ice to an analytical site that will perform the Pig-a analyses. Collect whole blood samples and combine with

kit-supplied Anticoagulant Solution, see Section 8.

Warning: Some capillary

tubes include a clot activator that will cause aggregation

and make the samples

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MutaFlow Rodent Blood Freezing Kit

Page 3 of 4 (Version 190508)

3. IMMEDIATELY after collection add 200 µL of blood to the bottom of an appropriately labeled EDTA tube and gently pipette up and down 3 times to mix with the Anticoagulant Solution.

4. Repeat Steps 2 and 3 for each rodent. Blood can be maintained in EDTA tubes at ambient temperature for up to 2 hrs. For longer periods of time, maintain EDTA tubes at 2 °C to 8 °C.

8.2. Blood Collection into Needle and Syringe 1. Collect blood.

a. If collecting blood with a small gauge needle and 1 cc syringe, it is important to first coat the inside of the needle/syringe with several hundred microliters of kit-supplied Anticoagulant Solution. Then, hold the needle/syringe upright and expel any air that may be in the barrel of the syringe. Proceed by discharging the Anticoagulant Solution from the syringe. For most needle and syringe combinations, this will leave behind the desired amount of Anticoagulant Solution in the so-called dead volume (approximately 50 to 60 µL). If using a fixed needle syringe with considerably less dead volume, it will be necessary to leave approximately 50 to 60 µL of Anticoagulant Solution behind.

b. Then, in order to achieve the proper ratio of Anticoagulant Solution to blood, your goal should be the collection of approximately 300 µL blood from each rodent. (Note that in the case of mice, this blood volume may exceed your institution’s maximal blood volume limit for a non-terminal bleed.)

2. Immediately after collection, transfer the entire volume of blood (approximately 300 µL) into the bottom of an EDTA tube (pre-aliquoted Anticoagulant Solution is not necessary in this case).

3. Repeat Steps 1 and 2 for each rodent. Blood can be maintained in EDTA tubes at ambient temperature for up to 2 hrs. For longer periods of time, maintain EDTA tubes at 2 °C to 8 °C.

9. Rodent Blood Freezing

1. Aliquot 500 µL of Rodent Blood Freezing Solution into pre-labeled cryovials, two per rodent.

2. Gently resuspend cells by pipetting and transfer 100 µL of whole blood/Anticoagulant Solution mixture from the EDTA tube into the Freezing Solution. Mix by gently pipetting 8 to 10 times. Repeat so that each sample is prepared in duplicate.

3. Repeat step 2 for additional samples in batches of up to 8.

4. Incubate each batch of up to 8 samples in Freezing Solution at room temperature for 5 minutes before transferring to a freezer set to –75 °C to –85 °C.

5. Samples should remain frozen for at least 48 hrs. They can be stored frozen for up to 1 year. Frozen samples can be analyzed for Pig-a frequencies by purchasing MutaFlow Rodent Blood Thawing Kit(s) and MutaFlowPLUS Kit(s), sold separately. Alternatively, frozen samples can be shipped to an analytical site for Pig-a analyses. In this case it is important for the samples to remain frozen throughout transit using dry ice, replenishing dry ice as necessary.

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10. References

§ Avlasevich SL, Phonethepswath S, Labash C, Carlson K, Torous DK, Cottom J, Bemis JC, MacGregor JT, and Dertinger SD (2014) Environmental & Molecular Mutagenesis 55, 400-406.

§ Bhalli JA, Ding W, Shaddock JG, Pearce MG, Dobrovolsky VN, and Heflich RH (2013) Mutation Research 753, 82-92. § Bhalli JA, Shaddock JG, Pearce MG, Dobrovolsky VN, Mutagenesis (2013) 28, 447-455.

§ Bhalli JA, Shaddock JG, Pearce MG, Dobrovolsky VN, Cao X, Heflich RH, and Vohr HW (2011) Environmental & Molecular Mutagenesis 52, 731-737.

§ Cammerer Z, Bhalli JA, Cao X, Coffing SL, Dickinson D, Dobo KL, Dobrovolsky VN, Engel M, Fiedler RD, Gunther WC, Heflich RH, Pearce MG, Shaddock JG, Shutsky T, Thiffeault CJ, and Schuler M (2011) Environmental & Molecular Mutagenesis 52, 721-730. § Dertinger SD, Avlasevich SL, Torous DK, Bemis JC, Phonethepswath S, Labash C, Carlson K, Mereness J, Cottom J, Palis J, and

MacGregor JT (2014) Toxicological Sciences 140, 307-314.

§ Dertinger SD, Bryce SM, Phonethepswath S, Avlasevich SL (2011) Mutation Research 721, 163-170.

§ Dertinger SD, Phonethepswath S, Avlasevich SL, Torous DK, Mereness J, Cottom J, Bemis JC, and MacGregor JT (2014) Environmental & Molecular Mutagenesis 55, 299-308.

§ Dertinger SD, Phonethepswath S, Franklin D, Weller P, Torous DK, Bryce SM, Avlasevich S, Bemis JC, Hyrien O, Palis J, MacGregor JT (2010) Toxicological Sciences 115, 401-411.

§ Dertinger SD, Heflich RH (2011) Environmental Molecular Mutagenesis 52, 681-684.

§ Dertinger SD, Phonethepswath S, Avlasevich SL, Torous DK, Mereness J, Bryce SM, Bemis JC, Bell S, Weller P, MacGregor JT, (2012) Toxicological Sciences 130, 328-348.

§ Dertinger SD, Phonethepswath S, Weller P, Avlasevich S, Torous DK, Mereness JA, Bryce SM, Bemis JC, Bell S, Portugal S, Aylott M, MacGregor JT (2011) Environmental Molecular Mutagenesis 52, 748-755.

§ Dertinger SD, Phonethepswath S, Weller P, Nicolette J, Murray J, Sonders P, Vohr H-W, Shi J, Krsmanovic L, Gleason C, Custer L, Henwood A, Sweder K, Stankowski LF Jr., Roberts DJ, Giddings A, Kenny J, Lynch AM, Defrain C, Nesslany F, van der Leede B-j, Van Doninck T, Schuermans A, Tanaka K, Hiwata T, Tajima O, Wilde E, Elhajouji A, Gunther WC, Thiffeault CJ, Shutsky TJ, Fiedler RD, Kimoto T, Bhalli JA, Heflich RH, MacGregor JT (2011) Environmental Molecular Mutagenesis 52, 690-698.

§ Dobo KL, Fiedler RD, Gunther WC, Thiffeault CJ, Cammerer Z, Coffing SL, Shutsky T, and Schuler M (2011) Mutation Research 725, 13-21.

§ Dobrovolsky VN, Miura D, Heflich RH, Dertinger SD (2010) Environmental Molecular Mutagenesis 51, 825-835.

§ Guérard M, Koenig J, Festag M, Dertinger SD, Singer T, Schmitt G, and Zeller A (2013) Toxicological Sciences 135, 309-316. § Graupner A, Instanes C, Dertinger SD, Andersen JM, Lindeman B, Rongved TD, Brunborg G, and Olsen A (2014) Mutation

Research 772, 34-41.

§ Lemieux CL, Douglas GR, Gingerich J, Phonethepswath S, Torous DK, Dertinger SD, Phillips DH, Arlt VM, and White PA (2011) Environmental & Molecular Mutagenesis 52, 756-765.

§ Lynch AM, Giddings A, Custer L, Gleason C, Henwood A, Aylott M, and Kenny J (2011) Environmental & Molecular Mutagenesis 52, 699-710.

§ Phonethepswath S, Avlasevich SL, Torous DK, Mereness J, Bemis JC, Macgregor JT, and Dertinger SD (2013) Environmental & Molecular Mutagenesis 54, 294-298.

§ Phonethepswath S, Franklin D, Torous DK, Bryce SM, Bemis JC, Raja S, Avlasevich S, Weller P, Hyrien O, Palis J, MacGregor JT, Dertinger SD (2010) Toxicological Sciences 114, 59-70.

§ Schuler M, Gollapudi BB, Thybaud V, Kim JH (2011). Environmental Molecular Mutagenesis 52, 685-689.

§ Shi J, Krsmanovic L, Bruce S, Kelly T, Paranjpe M, Szabo K, Arevalo M, Atta-Safoh S, Debelie F, LaForce MK, Sly J, and Springer S (2011) Environmental & Molecular Mutagenesis 52, 711-720.

§ Stankowski LF Jr, Roberts DJ, Chen H, Lawlor T, McKeon M, Murli H, Thakur A, and Xu Y (2011) Environmental & Molecular Mutagenesis 52, 738-747.

1. Materials Provided Additional Materials Required First-Time Users Ordering Information and Technical Services...

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