InhibiScreen Kinase Inhibitor Assay

Transcription

1 InhibiScreen Kinase Inhibitor Assay Flow Cytometric assessment of efficacy of PI3Kγ, PI3Kδ, BTK and SYK pathway inhibitors using the measurement of basophil activation in human EDTA and Heparin Whole Blood. For Research Use Only. Not For Use in Diagnostic Procedures. Catalog Number: Size: 81-INHBAT tests Version: November 29, G Keewaydin Drive, Salem, NH P: (800) F: (603)

2 INTENDED USE The Spectru InhibiScreen kit is intended for the flow cytometric investigation of the efficacy of inhibitors targeting the PI3K, BTK and SYK pathways via the analysis of CD63 antigen surface exposure on basophils in human EDTA and Heparin Whole Blood. PRINCIPLE OF THE ASSAY The Spectru InhibiScreen assay is based on the suppression of the in vitro stimulation of basophils in the presence of inhibitors of molecular targets such as PI3Kγ and PI3Kδ, SYK and BTK. This suppression is measured via subsequent flow cytometric analysis of externalized CD63 antigen expression on the basophil surface after degranulation. If there is sufficient quantity of the inhibitor of interest, basophil activation in the presence of IgE or fmlp is prevented. The exposed CD63 antigen is detected by monoclonal antibody staining (FITC labeled). The activated basophil population is identified by staining using the CCR3 monoclonal antibody (Rphycoerythrin (PE) labeled). A monoclonal antibody against the IgE receptor results in activation of the basophil via and immunological pathway which includes the PI3Kδ, BTK and SYK signaling pathways. Chemotactic peptide, N-formyl-Met-Leu-Phe (fmlp), activates basophils via PI3Kγ as well as other non-immunological signaling. MATERIALS SUPPLIED Cat. No. Component Quantity Preparation 900-IGE1 InhibiScreen Anti-IgE Stimulation Control 1 vial Lyophilized 900-FMLP1 InhibiScreen fmlp Stimulation Control 1 vial Lyophilized 900-SB1 InhibiScreen Stimulation Buffer 1 vial Lyophilized 900-SR1 InhibiScreen Staining Reagent 2 ml Ready to use 900-LS1 InhibiScreen Lysing Solution 25 ml 10X concentrated 900-WBB1 InhibiScreen Wash Buffer 50 ml Ready to use MATERIALS REQUIRED 5 ml test tubes for blood staining 96 well plate for blood staining Ultrapure demineralized water Automatic pipettes with disposable tips Vortex mixer Thermostat able to incubate test tubes or 96 well plate at 37 C Centrifuge with rotor suitable for test tubes or 96 well plate Flow cytometer-blue laser excitation at 488 nm, light emission at 525 nm (FITC) and 575 nm (PE) PRECAUTIONS 1. For Research Use Only. Not for use in diagnostic procedures. 2. Blood must be collected into a tube containing Heparin or K-EDTA. 3. Blood samples must be treated within 24 hours after collection if stored at 2-8 C. 4. Stained samples can be analyzed up to 96 hours after staining. Store at 2-8 C if not analyzed immediately. Page 2 of 7

3 5. The flow cytometer should be calibrated on a routine basis using fluorescent microbeads to ensure stable sensitivity of detectors. 6. Do not use reagents after expiration date stated on vial labels. 7. Avoid prolonged exposure of the InhibiScreen Staining Reagent to light. 8. Avoid contamination of reagents. 9. Any non-performance of assay procedure may product false results. 10. Blood samples are considered as potentially infectious and must be handled according to local regulations. 11. All materials derived from animal sources are bovine spongiform encephalopathy (BSE) negative. However, all materials should be kept from ruminating animals. 12. Avoid direct contact with skin. 13. This product is not for internal use. 14. Avoid eating, drinking, or smoking when using this product. 15. Do not pipette any reagents by mouth. 16. Reagents from this kit are lot-specific and must not be substituted. 17. Do not use reagents beyond the expiration date. 18. Variations to the test procedure are not recommended and may influence the test results. STORAGE CONDITIONS The kit should be stored at 2-8 C. The kit is stable until the expiration date on the box label. SAMPLE HANDLING Collect sufficient blood into K-EDTA or Heparin blood collection tubes. Perform the cell stimulation immediately or store the blood sample refrigerated (2-8 C) for up to 24 hours. Do not centrifuge or freeze blood samples. REAGENT PREPARATION All reagents must be equilibrated to room temperature prior to preparation and subsequent use in the assay. InhibiScreen Anti-IgE and fmlp Stimulation Controls are provided in a lyophilized form. Reconstitute using 1.1 ml distilled water. Close the vial with the rubber stopper and cap, gently swirl the vial prior to use. The contents of the vial should be in solution with no visible particulates. Unused volume of the reagent can be aliquoted, frozen once, and stored at 20 C for later use. Avoid repeated freeze/thaw cycles. InhibiScreen Stimulation Buffer is provided in a lyophilized form. Reconstitute using 10.5 ml distilled water. Close the vial with the rubber stopper and cap and gently swirl the vial. Unused volume of the reagent can be aliquoted, frozen once, and stored at 20 C for later use. Avoid repeated freeze/thaw cycles. InhibiScreen Lysing Reagent is provided in a 10X concentrated solution. Dilute with distilled water (e.g. 100 µl lysing reagent µl distilled water). 1X buffer can be stored at 2-8 C up to 5 days. InhibiScreen Staining Reagent and Wash Buffer are provided in a ready to use format. Page 3 of 7

4 ASSAY PROCEDURE Heparinized and EDTA whole blood have been validated for use with this assay. This assay may be performed in individual tubes or in a 96 well plate. Depending on your format of choice, refer to the specific assay procedure below. Incubation with inhibitor of interest (applies to both assay formats) 1. Mix blood aliquots with different concentrations of inhibitor of interest, or different compounds of interest dissolved in DMSO. Use volumetric ratio 1 vol. of DMSO + 99 vol. of blood. 2. Mix gently, and incubate mixtures for 60 minutes at 37 C. Assay Procedure for Individual Tube Format: Stimulation of inhibitor-treated/untreated blood samples 1. Add 100 L of Stimulation Buffer into all tubes. 2. Add 10 L of Stimulation Control of interest (anti-ige, fmlp) or Stimulation Buffer in case of negative control sample. 3. Add 20 L of Staining Reagent. 4. Add 100 L of inhibitor-treated (or untreated) blood sample. 5. Mix gently, and incubate tubes at 37 C for 15 minutes in water bath or 25 minutes in air incubator. Lysing 1. Add 2 ml of pre-warmed InhibiScreen Lysing Solution (1X concentrated) into all tubes, mix gently, and incubate for 5 minutes at room temperature. 2. Centrifuge tubes for 5 minutes at 300x g. 3. Remove the supernatant, resuspend the cell pellet with 300 µl of Wash Buffer and mix gently. Note: Depending on Flow cytometry instrumentation used, more wash buffer (e.g. 600 µl) may be necessary. Data Acquisition 1. Samples can be analyzed immediately, or stored at 2-8 C and analyzed within 96 hours. Assay Procedure for 96 Well Plate Format Prior to analysis, prepare the following cocktail for desired number of wells/plate. A total of 65 µl will be added to each well. Stimulation Buffer 50 µl/well + Staining Reagent 10 µl/well + Stimulation Control of Interest (anti-ige or fmlp) 5 µl/well Page 4 of 7

5 Stimulation of inhibitor-treated/untreated blood samples 1. Add 20 µl of inhibitor treated (or untreated) blood to each well 2. Add 65 µl of prepared cocktail to each well and pipette up and down to gently mix contents of well 3. Incubate at 37 C for minutes Lysing 1. Add 200 µl pre-warmed Lysing Solution (1X concentrated) to each well and gently mix by pipette. Incubate at room temperature 5-10 minutes. 2. Centrifuge 1250 RPM for 5 minutes without cooling. Discard supernatant. 3. Add 200 µl pre-warmed Lysing Solution (1X concentrated) to each well and gently mix by pipette. Incubate at room temperature for 5-10 minutes. 4. Centrifuge 1250 RPM for 5 minutes without cooling. Discard supernatant. 5. Add 200 µl Wash Buffer and gently mix by pipette. Data Acquisition 1. Samples can be analyzed immediately, or stored at 2-8 C and analyzed within 96 hours. FLOW CYTOMETRIC ACQUISITION Flow cytometric acquisition can be performed on any flow cytometer working with a 488 nm argon laser diode (blue-green excitation light). The flow cytometer must be equipped to detect forward scatter (FSC) and side scatter (SSC) and the two fluorochromes PE and FITC. Analyze stained samples using flow cytometer. In order to analyze sufficient number of basophils (>200), acquire 50, ,000 events per sample. Compensate your data (FITC and PE channels) prior to or after the acquisition. DATA ANALYSIS The analysis of the acquired data can be performed with any flow cytometry analysis software. Visualize compensated data as a dot-plot on the SSC versus fluorescence intensity in the PE channel. Figure 1a: Set the gate for basophil population (CCR3 positive, SSC low). The gate must be set individually for each sample. Figure 1b: Plot the gated basophils as a histogram where the X-axis represents fluorescence intensity in the PE channel. Set the gate for stimulated basophils (CD63 bright) using the positive control sample. Figures 1c and 1d: Calculate the percentage of activated basophils (CD63 bright) in the positive control stimulated samples versus the inhibitor treated samples. As the inhibitor dose is increased, the CD63 bright population decreases. Page 5 of 7

6 a. b. c. d. Figure 1 LIMITATIONS Blood sample may contain insufficient amount of basophils. Basophils in some blood samples are not susceptible to stimulation. Verify the preparations by eye to assess the efficacy of lysis. Flow cytometer may produce false results if the device has not been aligned and maintained properly. Data may be incorrectly interpreted if fluorescent signals were not correctly compensated or if gates were set inaccurately. Page 6 of 7

7 SHORT ASSAY PROTOCOLS INDIVIDUAL TUBE FORMAT 96 WELL PLATE FORMAT Mix Inhibitor with Whole Blood at a ratio of 1:100. Incubate for 60 min at 37 C. Mix Inhibitor with Whole Blood at a ratio of 1:100. Incubate for 60 min at 37 C. Mix Stimulation Buffer, Stimulation Control, Staining Reagent and Whole Blood at a ratio of 100/10/20/100. Mix gently and incubate for 15 min at 37 C. Mix Stimulation Buffer, Stimulation Control, Staining Reagent and Whole Blood at a ratio of 50/10/5/20. Mix gently and incubate for min at 37 C. Add 200 µl of pre-warmed Lysing Solution to all samples, mix gently and incubate for 5-10 min. Add 2 ml of pre-warmed Lysing Solution to all samples, mix gently and incubate for 5 minutes. Centrifuge for 5 minutes at 1250 rpm. Centrifuge for 5 minutes at 300g. Decant supernatant and resuspend in 200 µl of pre-warmed Lysis Buffer, mix gently and incubate for 5-10 min. Centrifuge for 5 minutes at 1250 rpm. Decant supernatant and resuspend in 300 µl Wash Buffer, mix gently. Decant supernatant and resuspend in 200 µl of Wash Buffer. Mix gently. Acquire on Flow cytometer immediately, or store at 2-8 C for acquisition within 96 hours. Acquire on Flow cytometer immediately, or store at 2-8 C for acquisition within 96 hours. Total Time = 60 minutes + 25 minutes Total Time = 60 minutes + 45 minutes Page 7 of 7