COMPONENT NAME COMPONENT # QUANTITY STORAGE SHELF LIFE FORMAT. Store at 2-8 C. Do not freeze. Store at 2-8 C. Do not freeze.

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1 This document is available at EasySep Anti-Rat IgGa Kit Catalog #8990 For processing x 0^9 cells Description EasySep Anti-Rat IgGa Kit contains all of the necessary components to create a customized EasySep selection cocktail when mixed with your own This selection cocktail can then be used to positively select cells with the cell surface antigen recognized by your rat IgGa monoclonal antibody. In addition to positive selection applications, selection cocktails from different EasySep Anti-Rat kits can be mixed and matched to make custom enrichment cocktails. Fast and easy-to-use Up to 99% purity No columns required Isolated cells are not fluorochrome-labeled Desired cells are labeled with antibodies and magnetic particles, and separated without columns using an EasySep magnet. Unwanted cells are simply poured off, while desired cells remain in the tube. Isolated cells are immediately available for downstream applications such as flow cytometry or cell culture. Component Descriptions COMPONENT NAME COMPONENT # QUANTITY STORAGE SHELF LIFE FORMAT EasySep Anti-Rat IgGa Component EasySep Dextran RapidSpheres C x 0. ml 000 x ml Store at - 8 C. Do not freeze. Store at - 8 C. Do not freeze. Stable until expiry date (EXP) on label. Stable until expiry date (EXP) on label. A combination of monoclonal antibodies in PBS with % HPCD. A suspension of magnetic particles in water. RoboSep Empty Vial 40 Not applicable Not applicable Not applicable HPCD - -hydroxypropyl-β-cyclodextrin; PBS - phosphate-buffered saline Components may be shipped at room temperature ( - C) but should be stored as indicated above. Sample Preparation BONE MARROW Flush bone marrow cells from femur and tibia into recommended medium using a syringe equipped with a gauge needle. Disperse aggregates by gently passing the cell suspension through the syringe several times. Alternatively, crush bones using a mortar and pestle. Remove remaining clumps and debris by passing cell suspension through a 0 µm mesh nylon strainer. Centrifuge at 00 x g for 0 minutes and resuspend cells at in recommended medium. SPLEEN Disrupt spleen in recommended medium. Remove aggregates and debris by passing cell suspension through a 0 µm mesh nylon strainer. Centrifuge at 00 x g for 0 minutes and resuspend at x 0^8 nucleated cells/ml in recommended medium. Ammonium chloride treatment is not recommended when preparing the cells for separation. Recommended Medium EasySep Buffer (Catalog #044), RoboSep Buffer (Catalog #004), or PBS containing % fetal bovine serum (FBS) and mm EDTA. Medium should be free of Ca++ and Mg++. Page of

2 EasySep Anti-Rat IgGa Kit Directions for Use Manual EasySep Protocols See page for Sample Preparation and Recommended Medium. Refer to Tables and for detailed instructions regarding the EasySep procedure for each magnet. Table. EasySep Anti-Rat IgGa Kit Protocol EASYSEP MAGNETS EasySep (Catalog #8000) The Big Easy (Catalog #800) Prepare sample at the indicated cell concentration within the volume range ml ml ml ( x mm) polystyrene round-bottom tube (e.g. Catalog #800) 4 ml ( x 9 mm) polystyrene round-bottom tube (e.g. Catalog #8008) Dilute your rat IgGa monoclonal antibody in your buffer of choice in a. ml polypropylene (microcentrifuge) tube. 0 µg/ml in buffer 0 µg/ml in buffer Prepare Selection Cocktail in a tube. For each ml of sample, prepare 0 µl of Selection Cocktail ( µl of Component + µl of rat IgGa monoclonal antibody). Mix equal volumes of Component and diluted Mix equal volumes of Component and diluted Incubate. RT for minutes RT for minutes 4 Add prepared Selection Cocktail to sample. 0 µl/ml of sample 0 µl/ml of sample 0 seconds 0 seconds Add RapidSpheres to sample. 40 µl/ml of sample 40 µl/ml of sample Add recommended medium to top up the sample to the indicated volume. Mix by gently pipetting up and down times. Top up to. ml Top up to ml for samples ml Top up to 0 ml for samples > ml Place the tube (without lid) into the magnet and incubate. RT for minutes RT for minutes 8 Pick up the magnet, and in one continuous motion invert the magnet and tube,* pouring off the supernatant. Remove the tube from the magnet; this tube contains the isolated cells. 9 Repeat steps as indicated. Steps and 8, three more times (total of 4 x -minute separations) Steps and 8, three more times (total of 4 x -minute separations) 0 Resuspend cells in desired medium. Be sure to collect cells from the sides of the tube. RT - room temperature ( - C) * Leave the magnet and tube inverted for - seconds, then return upright. Do not shake or blot off any drops that may remain hanging from the mouth of the tube. Page of

3 EasySep Anti-Rat IgGa Kit Table. EasySep Anti-Rat IgGa Kit Protocol EASYSEP MAGNETS EasyEights (Catalog #80) ml tube 4 ml tube Prepare sample at the indicated cell concentration within the volume range ml - 8 ml ml ( x mm) polystyrene round-bottom tube (e.g. Catalog #800) 4 ml ( x 9 mm) polystyrene round-bottom tube (e.g. Catalog #8008) Dilute your rat IgGa monoclonal antibody in your buffer of choice in a. ml polypropylene (microcentrifuge) tube. 0 µg/ml in buffer 0 µg/ml in buffer Prepare Selection Cocktail in a tube. For each ml of sample, prepare 0 µl of Selection Cocktail ( µl of Component + µl of rat IgGa monoclonal antibody). Mix equal volumes of Component and diluted Mix equal volumes of Component and diluted Incubate. RT for minutes RT for minutes 4 Add prepared Selection Cocktail to sample. 0 µl/ml of sample 0 µl/ml of sample 0 seconds 0 seconds Add RapidSpheres to sample. 40 µl/ml of sample 40 µl/ml of sample Add recommended medium to top up sample to the indicated volume. Mix by gently pipetting up and down times. Top up to. ml Top up to ml for samples ml Top up to 0 ml for samples > ml Place the tube (without lid) into the magnet and incubate. RT for 0 minutes RT for 0 minutes 8 Carefully pipette** (do not pour) off the supernatant. Remove the tube from the magnet; this tube contains the isolated cells. 9 Repeat steps as indicated. Steps and 8, two more times (total of x 0-minute separations) Steps and 8, two more times (total of x 0-minute separations) 0 Resuspend cells in desired medium. Be sure to collect cells from the sides of the tube. RT - room temperature ( - C) ** Collect the entire supernatant, all at once, into a single pipette (e.g. for EasyEights ml tube use a ml serological pipette [Catalog #800]; for EasyEights 4 ml tube use a 0 ml serological pipette [Catalog #8004]). Page of

4 EasySep Anti-Rat IgGa Kit Directions for Use Fully Automated RoboSep Protocol See page for Sample Preparation and Recommended Medium. Refer to Table for detailed instructions regarding the RoboSep procedure. Table. RoboSep Anti-Rat IgGa Kit Protocol RoboSep (Catalog #0000 and #000) Prepare sample at the indicated cell concentration within the volume range. Dilute your rat IgGa monoclonal antibody in your buffer of choice in a. ml polypropylene (microcentrifuge) tube. Prepare Selection Cocktail in the RoboSep Empty Vial provided. See Table 4 for required volumes. Incubate ml 4 ml ( x 9 mm) polystyrene round-bottom tube (e.g. Catalog #8008) 0 µg/ml in buffer Mix equal volumes of Component and diluted rat IgGa monoclonal antibody. RT for minutes 4 Select protocol. Anti-Rat IgGa 8990 Load the carousel. Start the protocol. Unload the carousel when the run is complete. Remove the tube containing the isolated cells and resuspend in desired medium. Be sure to collect cells from the sides of the tube. 0 seconds Follow on-screen prompts Press the green Run button Table 4. RoboSep Selection Cocktail Preparation START SAMPLE POSITIVE SELECTION COMPONENT RAT IgGA MONOCLONAL ANTIBODY PREPARED SELECTION COCKTAIL TOTAL VOLUME 0. ml. µl. µl µl ml µl µl 0 µl. ml 8. µl 8. µl µl ml 00 µl 00 µl 00 µl ml µl µl 0 µl 4 ml 0 µl 0 µl 00 µl ml µl µl 0 µl ml 00 µl 00 µl 400 µl ml µl µl 40 µl 8 ml 0 µl 0 µl 00 µl Note: RoboSep requires an extra 00 µl of the Selection Cocktail to run properly (compared to manual protocols). Page 4 of

5 EasySep Anti-Rat IgGa Kit Notes and Tips ASSESSING PURITY The rat IgGa antibody used in the cocktail created with EasySep Anti-Rat IgGa Kit may block other fluorochrome-conjugated antibodies that are used to assess purity by flow cytometry. Use fluorochrome-conjugated antibody clones that are not blocked by the rat IgGa antibody used in the selection cocktail. If this is not possible, one of the following methods can be used to assess purity: Add a fluorochrome-conjugated antibody at a concentration of 0. µg/ml immediately after adding the cocktail. This method labels the positive cells in the entire sample. Use alternative fluorochrome-conjugated markers, if applicable. Use a fluorochrome-conjugated secondary antibody, such as Goat Anti-Mouse IgG (H+L) Antibody, Polyclonal (Catalog #08). OPTIMIZING RECOVERY Recovery may be improved by increasing separation time in the magnet from to 0 minutes for each round. Recovery of positively selected cells is also dependent on the quality of the rat IgGa antibody used in the cocktail created with EasySep Anti-Rat IgGa Kit. Antibodies that have expired or that have been stored improperly may show lower affinity for the surface marker on the target cell, resulting in lower recovery. OPTIMIZING PURITY For samples with a desired cell starting frequency of less than 0 - %, additional separation rounds will likely improve purity. If desired, repeat Steps and 8 (Tables and ) an additional to times. Note that recovery will decrease with each additional round of separation. Also, for rare cells (i.e., cells representing less than % of the initial population), increasing initial cell concentration from to x 0^8 cells/ml may improve purity. OPTIMIZING PERFORMANCE In some cases, titration of the rat IgGa antibody and EasySep Dextran RapidSpheres may be required for optimal performance. The concentration of the rat IgGa antibody may be increased or decreased from the recommended 0 µg/ml to achieve optimal purity and recovery, and EasySep Dextran RapidSpheres may also be increased from 40 µl/ml, as needed. The volume of Component incubated with the chosen rat IgGa antibody can also be increased or decreased from the recommended : ratio. PREVENTING NONSPECIFIC BINDING To prevent nonspecific binding of antibodies to cellular FcR receptors, Normal Rat Serum may be added to samples at 0 µl/ml of cells. Data Flow cytometry analysis of mouse bone marrow cells processed with EasySep Anti-Rat IgGa Kit using a rat IgGa anti-mouse Ly-G antibody as the primary antibody for cell marker selection, and labeled with Anti-Mouse Ly-G Antibody, Clone A8, PE (Catalog #00PE). Histograms show labeling of bone marrow cells (Start) and isolated cells (Isolated). Labeling of start cells with Rat IgGa, kappa Isotype Control Antibody, Clone RTK8, PE (Catalog #00PE) is shown in the bottom panel (solid line histogram). STEMCELL TECHNOLOGIES INC. S QUALITY MANAGEMENT SYSTEM IS CERTIFIED TO ISO 48. PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED. Copyright 0 by STEMCELL Technologies Inc. All rights reserved including graphics and images. STEMCELL Technologies & Design, STEMCELL Shield Design, Scientists Helping Scientists, EasySep, EasyEights, RapidSpheres, and RoboSep are trademarks of STEMCELL Technologies Canada Inc. All other trademarks are the property of their respective holders. While STEMCELL has made all reasonable efforts to ensure that the information provided by STEMCELL and its suppliers is correct, it makes no warranties or representations as to the accuracy or completeness of such information. Page of