Protocols - Seahorse Bioscience
Contents
1. Well Glucose Oligomycin FCCP Rotenone Myxothiazol Note Vigorous mixing of the stock 20 uM oligomycin is required to prevent precipitation Rotenone and Myxothiazol are mixed together in the appropriate concentrations for injections www seahorsebio com 5 Notes Suggestions and Comments The methods described above have been used successfully with whole pancreatic islets isolated from both mouse and humans We believe that whole islets from other species can be used by following this protocol however the tissue species including age and sex and method of isolation will contribute to the overall activity and other variables associated with the whole islets Starting values ranges and optimaization it is recommended that the following parameters be explored and optimized depending on the overall goal s of the experiment and research topic e Amount of whole islets per well e The concentration of substrates and compounds injected e Mix Wait and Measure times References eeeeee e Please see Seahorase Biosciene s XF24 Trainig Course Workbook for a complete guide to operating and analyzing data used in the Seahorse XF24 Flux Analyzer Instrument For methods on isolating whole islets please see http www jove com video 255 murine pancreatic islet isolation Corporate Headquarters European Headquarters Seahorse Bioscience Inc Seahorse Bioscience Europe 16 Esquire Road Fruebjergvej 3 North Billeric2. 24 Islet Capture Microplate d Take care during this step that you don t cause too much turbulence so as to keep the islets resting in the depression at the bottom of the well Release the islet capture screen into the well by pulling up on the T lever on the capture screen inset tool Be sure the islet capture rings are stuck firmly at the bottom of the well This can be confirmed by gently pushing the screen down with a blunt pipette tip Figure 5 Make sure that there is an islet capture screen in each well even if there are no cells in the well A microplate without a full complement of screens will cause problems with the head on the XF24 unit 3 Run the Islet Capture Microplate on the XF24 a Place the microplate in an incubator set at 37 C without CO Store the microplate in the incubator for at least 1 h to equilibrate temp and adjust islet metabolism to 3 mM glucose While plate is incubating prepare cartridge with desired injections See step 4 After cartridge is filled with compounds for injection load the cartridge and start program and calibration When the XF24 calibration is complete place the islet plate into the XF24 Run the program After the program is complete you can normalize by counting the number of islets per well with the dissecting microscope Islets may also be harvested for further downstream analysis e g protein Some users have found that this step
3. Protocols Islet Assay using the XF24 Islet Capture Microplate Introduction XF Analyzers are most commonly used with an adherent monolayer of cells attached to an XF tissue culture plate Many researchers however desire to use more physiologic cellular sources such as primary pancreatic islets which are routinely utilized to study diabetes Seahorse Bioscience in collaboration with the Mitochondrial ARC Advancing Research through Collaborations at Boston University School of Medicine has developed a protocol that employs a novel consumable the XF24 Islet Capture Microplate to assess whole islet bioenergetics in vitro The advantages over traditional methods e g Clarke Electrode Apparatus include higher throughput 20 sample per assay and smaller amounts of pancreatic islets as low as 30 islets per well Materials and Methods The assay workflow Figure 1 describes the procedure used to prepare reagent materials and injected compounds The whole islet protocol described below is a modification of the XF24 Analyzer Protocol described in the XF24 User Manual Verson 1 Please feel free to modify the protocol to realize your intended research goals Modified XF Assay Media MA Media Supplement XF DMEM assay media with 3 mM NOTE Ahen planning ai anereate IIe glucose and 1 FBS to run whole islets FBS is needed to prevent the islets from becoming too adherent assay Seahorse recommends using an anti adherent for accurate re
4. a MA 01862 US 2100 Copenhagen DK Phone 1 978 671 1600 Phone 45 31 36 98 78 www seahorsebio com Seahorse Bioscience A part of Agilent Technologies Asia Pacific Headquarters Seahorse Bioscience Asia 199 Guo Shou Jing Rd Suite 207 Pudong Shanghai 201203 CN Phone 0086 21 33901768 201204308
5. a as TE 3 l described in Table 3 Measure 3 Table 3 Dilutions of Modified XF Assay Media Mix 2 Wait 2 Catalog Final ae Compound Brand Number Eancentration Dissolve in Measure 3 Mix 2 Stock 1000X in DMSO MENENG sigma naels Dilute to 10X in MA Media Wait 2 Stock 1000X in DMSO Measure 3 Cel elt Sula G Dilute to 10X in MA Media Stock 1000X in DMSO LS l oc in FOGE sigma ee Dilute to 10X in MA Media Mix 2 Stock 1000X in DMSO Wait D Glucose agma I Dilute to 10X in MA Media Measure 3 FBS MP Biomedicals 155765 Methanol Mix 2 Note Oligomycin FCCP rotenone and myxothiazol should be freshly diluted in MA Media for Wait 2 each experiment Stock solutions in DMSO may be stored at 20 C Measure 3 Mix 2 Other items needed Wait 2 XF24 Sensor Cartridge Measure 3 l Inj B XF24 Islet Capture Microplate deed Mix 2 Islet Capture Screens Wait 2 Capture Screen Insert Tool Measure 3 Mix 2 Calibration buffer Seahorse Bioscience Wait 2 Dissecting microscope Nessie 3 Open faced bio hood Mix 2 Wait 2 Multi channel pipettes and tips Measure 3 Eppendorf and 15 50 ml Falcon tubes Default equilibrate command consists of 2 min Mix 2 min Wait repeated 3X The same pattern Day Before the Assay could be followed for more injections 1 Prepare an XF Assay Template via the Assay Wizard a Using the XF24 Operation Manual as a guide and incorporating proper experimental design b Upload the assay template to the XF24 Analyzer be
6. esponse to glucose addition as clonal INS1 beta cells run in a standard XF culture plate Red lines Blank injection at A oligomycin at B Blue lines 20 mM glucose injection at A oligomycin at B Unpublished data from the Shirihai lab at Boston University School of Medicine Tables 7 amp 8 show a direct camparison between normal human islets and diabetic human islets Note that the basal OCR readings for the normal islets are 4X higher than that of diabetic islets and the response to glucose is depressed in the diabetic islets as compared to the normal islets Table 7 Oxygen Consumption Rate vs time for Normal Human Islets Table 8 Oxygen Consumption Rate vs time for Diabetic Human Islets OCR vs TIME OCR OCR vs TIME Avg OCR ab 292 A B 821 A B 263 736 233 651 204 566 481 174 144 N rp g 396 E 5 an g s X am Opor S 8 142 52 57 26 28 3 7 8 19 4 30 9 424 53 9 65 5 770 88 5 100 0 123 1 24 9 32 6 40 3 48 0 55 7 63 5 71 2 78 9 86 6 94 3 109 7 125 1 140 6 156 0 171 4 TIME min TIME min Comparisons of normal human islets versus diabetic human islets run in the Islet Capture Microplate Red lines Blank injection at A oligomycin at B Blue lines 20mM glucose injection at A oligomycin at B Unpublished data from the Shirihai lab at Boston University School of Medicine Table 4 XF sensor cartridge injection port compounds table Injection Ports Volume Concentration in Port Final Concentration in
7. fore starting the assay The experiment outlined here is an example of how to obtain the various mitochondiral respiration states using the XF24 c Use Table 3 as a guide to program the Mix Wait Measure and Injection protocol 2 Prepare the XF Sensor Cartridge a Hydrate the XF Sensor Cartridge overnight in XF Calibration Buffer at 37 C without CO b Prepare whole islets by the standard protocol s used in your laboratory For the protocol described here 8 mice were sacrificed to obtain 1400 islets enough for 20 wells at 70 islets well Incubate whole islets in a petri dish overnight under standard conditions for islet culture For the data shown islets are cultured in RPMI media with 11 mM glucose 10 FBS and 1 pen strep 2 www seahorsebio com Day of the Assay 1 Add whole islets and capture screens to the wells C Aspirate islets from petri dish and dispense into a 50 ml tube a b Wash 1X in MA Media Remove supernatant and re suspend in 2 ml MA Media While creating turbulence in the tube with a 20 ul pipettor take 20 ul aliquots and place as a drop ona culture dish make 3 drops total this gives you 3 of the islets Count islets under a dissecting microscope This will give you an average amount of islets per volume from which you can estimate the total number of islets Determine the count of the islets and adjust volume so you get 70 islets for every 100 ul of medial 700 is
8. lets ml Add 400 ul MA Media to each well of the XF24 Islet plate h Add 50 ul of the islet suspension to each well and repeat so each well gets a total of 100 ul of the islet suspension Final volume should be 500 ul per well When islets are seeded use a 20 ul pipette to move all of the islets ionto the depressed chamber in the bottom of the well This step is tedious use a dissecting microscope to be sure all of the islets are in the depression at the bottom of the well as in Figure 2 2 Add screens by pre wetting them in MA Media in a small Petri dish to remove any air bubbles See Figure 3 a Use a pair of sterile forceps to position the screens so that the ring is facing up Figure 3 Use the capture screen insert tool to pick up an islet capture screen from the petri dish Carefully place the islet capture screen in the bottom of each well using the capture screen insert tool Figure 4 Islet Assay using the XF24 Islet Capture Microplate Figure 3 Steps outlined in Day of Assay Step 3 Face up Pre wetting of screens to remove air bubbles Use capture screen Insert Tool Press firmly into place to capture screen Screen Capture 00 Head Screen t Figure 2 Be sure all of the islets are in the depression at the bottom of the well Cells Move islets into the depression at the bottom of the well www seahorsebio com 3 Islet Assay using the XF
9. producable results Please contact Seahorse Technical Support with any questions J E E E E E E O O E O E O O O O E E O E O E E E O O E E O E E E E E E O E O O E E E E E E E O E E E E O E E E E E a E E E O E E E E E O E E E E E E a E a E E Figure 1 Pancreatic Islet Assay Workflow Day Before Assay Day of Assay Add the islets into Warm plate at 37 C the appropriate wells of for 1 hr Transfer the islet capture microplate plate to XF24 Analyzer DY ees ee upon calibration completion sF a md es E AT fmia a a Dilute compunds into Modified Assay Medium MAS at 10X the Transfer the islets from desired final the outer shelf to the concentration inner depressions Run experiment Add to injection ports Place the islet capture n of cartridge screens into the wells SON ETE NE using the capture screen islet tool Analyze Data Yeahorse Bioscience A part of Agilent Technologies Islet Assay using the XF24 Islet Capture Microplate Table 2 Components Formulation of Modified XF Assay Media Table 3 Typical Mix and Measurement Cycle times for XF24 3 assays Gamcound Brand Catalog MW or Molar Final Grams or ml for 500 ml p Number Concentration Concentration of XF Assay Media Time Command minutes Glucose Sigma G7528 180 Calibration FBS Hyclone SH30070 03 100 Equillibrate l _ l l l Mix 2 It is recommended that all compounds to be added or injected are diluted into MA Medi
10. was not necessary as basal rates were sufficient for normalization 4 Prepare Biosensor Cartridge with Injections and Calibrate a b e 0o 0o oo o Before calibration load the XF sensor cartridge injection ports with following compounds listed in Table 4 Next page bottom Calibrate the sensor cartridge loaded with desired compounds as described in the XF manual eeeeeeeecseee eseoeesececes3seeeeesese e eese3 s5ee www seahorsebio com Figure 4 Day of Assay Place Islet Capture Screen Carefully place the screen in the well create as little turbulence as possible At Pull up on the T lever to release the capture screen once it s in place Figure 5 Day of Assay Checking the screen placement Well with screen in place Islets under screen Islet Assay using the XF24 Islet Capture Microplate Data Analysis The results in tables 5 amp 6 were obtained using 70 mouse islets well or isolated beta cells Table 5 Oxygen Consumption Rate vs time for Whole Islets Table 5 Oxygen Consumption Rate vs time for Beta Cells OCR vs TIME OCR vs TIME OCR OCR 570 570 r A B 508 508 446 446 385 _ 385 lt E 323 323 op op 2 ce O o 261 261 S amp T 199 g 18 S OG O 4137 O 4137 76 76 14 14 48 48 15 8 28 9 42 0 551 681 81 2 94 3 1074 120 5 133 5 146 6 O 7 14 20 27 34 41 48 54 61 68 75 82 88 95102 116 129 TIME min TIME min Whole pancreatic islets show a similar r
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