A High-content In Vitro Pancreatic Islet β-cell Replication Discovery Platform

Podsumowanie

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet β-cell replication and to develop methods for stimulating β-cell regeneration. Herein a high-content screening method to identify and assess the β-cell replication-promoting activity of small molecules is presented.

Streszczenie

Loss of insulin-producing β-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet β-cells remains an attractive strategy. β-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain β-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify β-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a β-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on β-cells and the molecular mechanisms that regulate β-cell growth.

Wprowadzenie

Diabetes encompasses a collection of disorders sharing the common end-point of disrupted glucose homeostasis. Although the pathogenic mechanisms of diabetes subtypes are distinct, they share the consequence of decreased β-cell mass, i.e., loss of insulin production capacity^1,2. Presently, diabetes treatment strategies rely upon chronic administration of exogenous insulin, pharmacologic stimulation of insulin production or enhancement of insulin sensitivity, and rarely, the transplantation of pancreatic islets or whole pancreas^3,4. Regrettably, the success of these strategies is short-lived and/or fails to sufficiently recapitulate the function of endogenous insulin production. Despite the utility of developing a method to stimulate β-cell regeneration, no such approach exists. Consequently, a major diabetes research goal is to develop methods to generate new β-cells or to expand endogenous β-cell mass⁵. Although β-cell regeneration from renewable sources such as embryonic stem cells is advancing, safety and efficiency concerns make the pursuit of alternative strategies, including expansion of mature β-cells, a priority^6,7. Importantly, the predominant source of new β-cells in vivo is pre-existing β-cells rather than specialized progenitor cells^8,9. Although β-cells appear to have limited replication capacity, a small increase in β-cell mass (~ 30%) may be sufficient to restore glucose homeostasis in many diabetics. Furthermore, in situ pharmacologic stimulation of β-cell mass is a potentially inexpensive and scalable treatment strategy. Herein a high-content screening method for identifying and characterizing small molecules that stimulate β-cell growth is presented.

A variety of in vitro experimental methods may be used to identify gene products and/or molecules that promote primary β-cell replication. Early efforts for measuring β-cell replication induction used fetal rodent pancreata culture or intact isolated islet cultures to measure [³H] thymidine incorporation, BrdU incorporation or mitotic bodies within the aldehyde-thionine or insulin stained population in response to specific treatment conditions^10,11. These in vitro approaches and close variants thereof have several limitations. Prominent deficiencies include (1) the use of fetal cells which, unlike mature β-cells, display a high basal β-cell replication rate and are growth regulated in a distinct manner¹²; (2) the subjective nature of experimenter-dependent adjudication of β-cell replication events; (3) the labor and time intensive nature of experimenter-dependent counting of β-cell replication events retards experimental throughput; (4) the use of nuclear incorporation/stain/appearance to identify replication events and a non-overlapping cytoplasmic stain to identify β-cells leads to misattribution of proximate non-β-cell replication events to β-cells.

More recently mature primary β-cells have been used to assess the impact of transgene over-expression as well as gene product or compound treatment on β-cell replication^13-16. However, these studies have also relied upon subjective counting of replication events, cytoplasmic staining- or non-specific methods for β-cell identification and/or labor-intensive steps that limit throughput, e.g., individual slide-well plating of cells or intact islet paraffin embedding and processing¹⁷. Notably, an image-based human β-cell replication screening methodology, similar to the one presented herein, has been published¹⁸; however, successful use of this assay has not been demonstrated and the use of human islets for primary screening may not be broadly feasible.

An alternative strategy for identifying replication-promoting substances is to assess growth induction of β-cell lines. Initial efforts used transformed β-cell-lines such as min6 cells or INS 832/13-cells^14,19-21. However, these cell-lines demonstrate unrestrained growth and bear little resemblance to well-differentiated β-cells²². Consequently, growth-induction capacity is minimal, of unclear relevance and sometimes difficult to recapitulate. An improved strategy for cell-line based screening utilizes "reversibly transformed" cells that are growth arrested in the absence of tetracycline (doxycycline)-dependent SV40 T antigen expression^23,24. However, it is unclear whether these cells revert to a "normal" β-cell-like state upon doxycycline removal. Unfortunately, use of these cells has yielded generalized growth-promoting compounds that don't appear to have immediate utility²⁴. Overall, the use of cell-lines to study growth regulation of a cell-type displaying minimal spontaneous replication activity may have limited applicability.

The β-cell replication screening platform presented herein utilizes mature primary rat β-cells to retain in vivo growth regulation to the extent possible, islet-cell cultures of mixed cell-type composition to enable identification of lineage-restricted growth-promoting activities, multi-well formatting to maximize throughput and automated analysis to eliminate bias and facilitate throughput. Successful use of this platform has enabled identification of several compounds that promote β-cell replication^25,26. Additionally, the assay has been used for structure-activity relationship studies and chemical epistasis experiments to provide mechanistic insights into the molecular regulation of β-cell replication. The presented platform was successfully adapted for lentiviral RNAi-based investigation of β-cell replication pathways²⁵. Limitations of the assay include restricted scalability (use of primary cells), use of rodent rather than human islet-cells (though the assay may be adapted for human islet studies), expenses associated with antibody-based imaging and primary islet use, the use of dispersed islets (disrupted islet architecture) to facilitate automated image acquisition and dependence upon the availability of an automated microscope with image acquisition and analysis capability. Although a facile in vivo screening methodology for identifying gene products or compounds that stimulate β-cell regeneration in situ would be ideal, such a platform is not yet available²⁷. Consequently, the described platform is appropriate for researcher interested in investigating most aspects of β-cell replication.

Protokół

This protocol was carried out in accordance with the Institutional Animal Care and Use Committee (IACUC) of Stanford University School of Medicine. The described protocol is scaled for islet isolation from six 250 - 300 g (8 - 9 weeks old) male Sprague Dawley rats, which is sufficient to generate 228 wells of a 384-well plate for islet-cell replication assessment.

1. Material Preparation

1. Prepare coating media prior to initiating islet isolation by collecting the conditioned media of 804G rat bladder carcinoma cells maintained at confluence for 3 days (20 ml of RPMI1640) in a 15 cm tissue culture dish ²⁸. Sterile filter and store (-20 °C) the conditioned media for later use.
2. Sterilize surgical tools (one 12 cm toothed tissue forceps, one 11.5 cm fine scissors, one 14.5 cm surgical scissors, two 16 cm curved forceps, one 12 cm curved hemostat, one 12 cm scalpel handle) and one 30 mesh tissue sieve prior to initiating islet isolation.
3. Prepare pancreatic digestion solution by dissolving a 60%:40% mixture of purified class I:class II collagenases (total collagenase activity of 300,000 - 400,000 units) in 60 ml of 1x Hanks' Balanced Salt Solution (HBSS) supplemented with calcium and magnesium. Load 10 ml of pancreatic digestion buffer into 10 ml syringes (six) with a 1" 22 G needle and place on ice.
   Note: 10 ml of digestion solution is injected per rat. Scale accordingly.
4. Prepare 4.5 ml of anesthetic cocktail in a ventilated hood by mixing 1.3 ml of ketamine (100 mg/ml), 0.2 ml of xylazine (100 mg/ml) and 3 ml of PBS. Prepare the anesthetic cocktail within 1 hr of initiating islet isolation.
5. Prepare wash buffer by adding 25 ml of newborn calf serum to 500 ml of HBSS. Place wash buffer on ice for later use.
6. Prepare 1 bottle of islet medium which is 500 ml of low glucose Dulbecco's Modified Eagle Medium (DMEM), 50 ml fetal bovine serum (FBS), 5,000 units/ml of penicillin and 5,000 µg/ml of streptomycin.
7. Prepare islet function medium from functionality/viability solution (500 ml) with 2% FBS, 2 mM glutamine, 5 mM glucose, 5,000 units/ml of penicillin and 5,000 µg/ml of streptomycin. Store the islet function media (4 °C) for later use.
8. Prepare 40 ml blocking buffer by adding 2.5 ml donkey serum and 0.12 ml triton X-100 to 37.38 ml of 1x phosphate buffered saline (PBS). Store blocking buffer (4 °C) for later use.
9. Obtain the required antibodies prior to initiating the protocol.
   Note: PDX-1 (TRITC) and Ki-67 (FITC) co-staining is used for primary β-cell replication screening. For lineage-specific replication analysis, perform immunofluorescence staining for additional cell identity markers (insulin, glucagon, vimentin or somatostatin; TRITC) along with nuclear (DAPI), PDX (Cy5) and Ki-67 (FITC) staining. Alternative replication markers, e.g., PCNA, may also be used.
10. Prepare a 228-well treatment plan with each treatment condition performed in quadruplicate. Include positive- (5-Iodotubercidin [1 µM] or dipyridamole [15 µM]) and vehicle-control (DMSO 1:666 dilution) wells.

2. Perfusion of the Pancreas

1. Anesthetize rats by i.p. injection of diluted anesthetic cocktail solution (0.30 ml/100 g body weight). Once the rat is fully anesthetized and unresponsive to noxious stimuli, perform cervical dislocation.
2. Place the euthanized rat in supine position (cranial-caudal orientation) on a stack of paper towel and spray the abdominal area with 70% ethanol.
3. Open the abdominal cavity with a U-incision (base at pubic region) and retract skin in the rostral direction over chest to expose the abdominal cavity.
4. Carefully grab the duodenum using two pairs of curved forceps, locate the duodenal entry of the common bile duct (sphincter of Oddi) and clamp the duodenal opening at the papilla with a curved hemostat.
5. Lift the curved hemostat used to clamp the common bile duct papilla and grab the bile duct close to liver using a curved forceps. Use blunt dissection with the curved forceps to isolate and expose the bile duct.
6. Reposition the rat with the head proximal and the feet distal.
7. Cannulate the common bile duct (CBD) at or just distal to the junction of the cystic and common hepatic ducts with a pancreatic digestion solution-loaded 10 ml syringe and slowly inject 10 ml of the pancreatic digestion solution. While injecting, support the CBD with a scalpel handle. Reposition the needle if the duct and pancreas fail to inflate.
8. Remove the inflated pancreas using the curved forceps and fine scissors to separate it from the descending colon, intestines, stomach and spleen. Place the inflated pancreas on ice in a 50 ml tube containing 5 ml of pancreatic digestion solution.
   Note: For maximum islet yield, collect all pancreata within 30 min and place only 1 or 2 pancreata in each 50 ml digestion tube.

3. Dissociation of the Pancreas

1. Once all pancreata are collected, place the digestion tubes into a 37 °C water bath for 15 min. Gently swirl the tubes every 3 min.
2. After 15 min, vigorously shake (vertically) the tubes 10 times and add 10 ml of cold wash buffer. Vigorously shake the tubes an additional 5 times and place on ice.
3. Fill the digestion tubes to 50 ml with cold wash buffer and mix by inverting tubes 5 times.
4. Centrifuge the tubes at 97 x g at 4 °C for 1 min and pour off the supernatant. Be careful not to dislodge the pelleted tissue.
5. Add 25 ml wash buffer and re-suspend the tissue by gently vortexing. Repeat steps 3.4 and 3.5 twice more.
6. Place a 30 mesh tissue sieve over a sterile 250 ml beaker and pour the tissue suspension onto the sieve. Rinse the digestion tube with an additional 20 ml of wash buffer and pour onto the mesh. The undigested pancreatic and fat tissues are removed at this step.
7. Pour the filtered material into two fresh 50 ml tubes and pellet the tissue by spinning at 97 x g for 1 min at 4 °C. Decant supernatant and invert tubes to drain excess buffer.

4. Purification of Islets

1. Add 20 ml of cold polysucrose/sodium diatrizoate solution (1.119 g/ml density) to the pelleted pancreatic tissue. Homogenously re-suspend the contents of each tube by gently pipetting up and down five times.
2. Gently overlay the polysucrose/sodium diatrizoate solution with 10 ml HBSS. Maintain a sharp liquid interface by slowly adding the HBSS along the tube wall.
3. Centrifuge the digested pancreata at 560 x g for 15 min (4 °C) with slow acceleration and no brake.
4. Collect the islet layer from the interface using a 10 ml pipette and place islets in fresh 50 ml tubes. Do not pool islets at this stage.
5. Add 40 ml of wash buffer to each 50 ml tube and spin for 1 min at 140 x g at 4 °C.
6. Pour off the supernatant and re-suspend pooled islets in 50 ml of wash buffer by inverting the tubes several times. Centrifuge the tubes for 1 min at 97 x g at 4 °C to collect islets in a pellet.
7. Pour off the wash buffer and repeat the wash. Bring islets into the tissue culture hood and aspirate the supernatant.
8. Re-suspend each tube of islets in 6 ml of islet medium.
9. Transfer the islets from each 50 ml tube into a well of six-well tissue culture dish. Swirl the 6-well dish to collect islets into the middle of the well and observe islet quality and purity under a microscope.
10. Manually collect islets using a 1 ml pipette and transfer the picked islets into the adjacent well containing 6 ml of islet media. Repeat islet swirling and picking until > 90% purity is achieved.
11. Transfer the purified islets to a 10 cm tissue culture dish containing 20 ml of islet medium.
12. Place islets in a tissue culture incubator (37 °C, 5% CO₂) overnight (Figure 1A).
13. In preparation of plating islet cells, coat the wells of a 384-well plate with 40 µl of 804G conditioned media per well. Incubate overnight in a tissue culture incubator.

5. Dispersion and Plating of Islet Cells

1. The following day, gently detach islets from the 10 cm dish with a cell scraper and transfer the islets into a 50 ml tube.
2. Pellet the islets by centrifugation for 1 min at 22 x g at room temperature. Aspirate the supernatant.
3. Wash the islets with 20 ml of warm PBS and decant as above.
4. Re-suspend islets in 0.25 % trypsin (150 µl per rat pancreas) and incubate at 37 °C for 10 min. Pipette islets up and down 10 times using 1 ml pipette after 5 and 10 min of incubation.
5. After 10 min, evaluate a 20 µl sample of the trypsinized islets under the microscope to ensure complete digestion and to count the islet cells using a hemocytometer. If undigested islets remain, continue incubation for an additional 3 - 5 min and pipette up and down 10 more times. Repeat as necessary.
   Note: The typical yield is ~ 125,000 - 150,000 islet-cells per rat.
6. Remove 804G conditioned media-coated 384-well plate from the incubator and remove media using a 12-well manifold aspirator.
7. Suspend islet cells at a density of 45,000 cells per ml in Islet function medium and plate 70 µl (3,150 cells) per well with a multichannel pipette (Figure 1B).
   Note: Because β-cells located in the outer wells tend to migrate towards the perimeter of the plate use rows C to N and columns 4 to 21 to plate 228 wells with the islet-cells isolated from 6 rats.
8. Place the plate in the tissue culture incubator for 48 hr to allow cell adhesion.

6. Islet-cell Culture Treatment, Fixation and Staining

1. Prepare 200 µl of vehicle and compounds (treatment conditions are performed in quadruplicate) at a 2x concentration in Islet function medium. Arrange compounds in a sterile 96-well plate to facilitate multi-well pipetting.
2. Carefully remove islet medium with a 12-well manifold aspirator and replace with islet function medium (35 µl/well) using a 12-well pipette. Remove and replace media from one row at a time to limit risk of drying (Figure 1C).
3. Initiate treatment by transferring 35 µl/well of the 2x compound solutions. Return plate to the incubator for the 48 hr treatment duration.
4. After 48 hr of treatment, decant the treatment media by inverting the plate.
5. Gently wash the islet cells with 50 µl per well of 1x PBS (1 min). Add the PBS using a multi-channel pipette.
6. Decant the PBS and fix the cells by adding 50 µl per well of cold 4% paraformaldehyde (PFA) diluted in 1x PBS. Incubate cells for 15 min at 4 °C.
7. Wash the cells three times (5 min each) by inverting the plate to remove the PFA and gently adding 60 µl of PBS per well as above.
8. Prepare 15 ml antigen retrieval solution by mixing 14.25 ml formamide and 0.75 ml 0.15 M sodium citrate (pH 6). Make the antigen retrieval solution immediately before use.
9. Remove PBS from wells by inverting the plate and add 60 µl antigen retrieval solution to each well. Heat the plate in 70 °C water bath for 45 min. Place a heating block on top of the plate during this incubation to prevent warping of the multi-well plate.
   Note: The water should be half-way up the plate skirt; the plate should not be submerged.
10. Remove the plate from water bath and allow it to cool for 20 min at room temperature. Ensure that the plate is not warped by visual inspection.
11. Wash the cells with 60 µl per well of 1x PBS three times (5 min each). Add PBS with a multi-channel pipette and decant PBS by inverting the plate.
12. Use a multi-channel pipette to add 40 µl per well of blocking buffer and incubate for 30 min at room temperature. Remove the blocking buffer by inverting the plate and decanting.
13. Add 40 µl of mouse anti-human Ki-67 (1:200) and goat anti-human PDX-1 (1:100) antibodies diluted in blocking buffer to each well and incubate at 4 °C overnight.
14. The next day, decant the anti-body solution and wash the cells with 1x PBS three times (5 min each) as described above. Decant the PBS.
15. Add 40 µl per well of diluted (1:200) biotinylated donkey anti-mouse IgG and rhodamine-conjugated donkey anti-goat IgG in blocking buffer. Incubate for 1 hr at room temperature.
16. Wash the cells with 1x PBS three times, 5 min each. Add 40 µl of diluted (1:400 in blocking buffer) fluorescein-conjugated streptavidin to each well. Incubate 30 min at room temperature.
17. Wash the cells with 1x PBS three times, 5 min each. Add 40 µl per well of DAPI (300 nM in blocking buffer) and incubate for 15 min at room temperature.
18. Wash cells with 1x PBS two times and leave cells in 40 µl 1x PBS. The plate is now ready to be analyzed.

7. β-Cell Replication Analysis

Note: An assay protocol must be established for the high content screening microscope used to measure β-cell replication. In its simplest composition, this protocol is a two-color assay where an identity marker is used to define β-cells (PDX-1⁺-cells) and a replication marker (Ki-67) is used to define cell division events.

1. Identify objects (β-cells) based upon the average fluorescence intensity of PDX-1 staining (549 nm filter) within an approximate circle (length:width < 1.6) within the expected pixel area of a β-cell nucleus (Figure 2A).
2. Next, establish settings to identify replication events (Ki-67-positivity) based upon the average fluorescence intensity (485 nm filter) within the PDX-1⁺ area (Figure 2B).
   Note: Exposure times must be fixed to allow pixel intensity comparisons across images. Assay protocol parameters are adjusted such that machine calls consistently exclude nonspecific staining, debris and cell aggregates that could adversely affect data quality.
3. Analyze a minimum of 500 β-cells per well to maximize accuracy and limit variability.
   Note: The basal β-cell replication index in rat islet cultures is expected to be 0.5 - 3%. Typically, 40 images per well is more than sufficient to identify 500 β-cells.
4. Prior to analyzing the entire plate, analyze selected negative- (DMSO-treated) and positive-control (5-iodotubercidin [1 µM] or dipyridamole [15 µM]) -treated wells to assess the validity of the assay protocol. When the assay is established correctly, positive control compounds induce at least a two-fold increase in the percentage of replicating β-cells.
5. Read the entire plate once the assay protocol is validated.

Wyniki

To assess β-cell or α-cell replication, a four-color assay protocol is required. First, objects are identified by DAPI staining (Channel 1, 386 nm). Next, β-cells (event 1) are counted: objects that co-express PDX-1⁺ (channel 2, 650 nm) and peri-nuclear insulin (channel 3, 549 nm). Subsequently, replicating β-cells (event 2) are counted: β-cells (event 1) that co-express Ki-67 (channel 4, 485 nm) (Figure 3). The percentage of replicatin...

Dyskusje

Experimental methods for studying the molecular pathways that control β-cell growth and regeneration are important tools for diabetes researchers. Herein, a rat-islet-based screening platform to identify and characterize small-molecule stimulators of β-cell replication is presented.

While most aspects of this protocol are easily performed by experienced researchers, a few steps require particular technique. First, during islet isolation, cannulation of the bile-duct without disruptin...

Materiały

Name	Company	Catalog Number	Comments
250 g male Male Sprague Dawly Rat	Charles River	Stain # 400
12 cm teeth tisuue forceps	Fine Science Tools	11021-12
11.5 cm fine scissors	Fine Science Tools	14058-11
14.5 cm surgical scissors	Fine Science Tools	14001-14
16 cm curved forceps	Fine Science Tools	11003-16
12 cm curved hepostat	Fine Science Tools	13011-12
12 cm scalpel handle	Fine Science Tools	10003-12
Tissue sieve-30 mesh	Bellco Glass	1985-85000
Cizyme RI, 375,000 CDA units	VitaCyte	005-1030
Hanks' Balanced Salt solution (Ca++ and Mg++)	Gibco	24020-117
Ketamine HCl (200 mg/20 ml)	JHP Pharmaceuticals	NDC# 42023-113-10	to make anesthetic cocktail
Xylazine (5 g/50 ml)	LLOYD	NADA# 139-236	to make anesthetic cocktail
Histopaque 1077	Sigma	H-1077	to make histopaque 1100
Histopaque 1119	Sigma	H-1119	to make histopaque 1100
Newborn Calf Serum 500 ml	Hyclone	SH30118.03
Hanks' Balanced Salt solution	Hyclone	SH30268.01
Dulbecco's Modified Eagle Medium/Low Glucose	Hyclone	SH30021.01
Functionality/Viability Solution	Mediatech	99-768-CV
RPMI1640 media	Hyclone	SH30096.01	to make conditioned medium
804G rat bladder carcinoma cell-line	Available upon request		to make conditioned medium
Fetal Bovine Serum, Qualified	Gibco	26160
GlutaMax-I	Gibco	35050-061
Penicillin (5,000 IU/ml/Strptomycin (5 mg/ml)	MP Biomedicals	1670049
Formamide 500 ml	Fisher BioReagents	BP227-500
Antigen Unmasking Solution 250 ml (pH 6.0)	Vector Laboratories	H-3300	to make 0.15 M Sodium Sitrate solution
Dextrose, Anhydrous	EMD Chemicals	DX0145-1	to make 1 M glucose solution
Nomal Donkey Serum (Powder)	Jackson ImmunoResearch	017-000-121
Triton X-100	Sigma	T8787-100ML
Mouse anti-human Ki67 antibody	BD Biosciences	556003
Goat anti-human PDX-1 antibody	R&D Systems	AF2419
Polyclonal Guinea Pig anti-insulin antibody	Dako	2016-08
Polyclonal Rabbit anti-glucagon antibody	Dako	2014-06
Polyclonal Rabbit anti-somatostatin antibody	Dako	2011-08
Polyclonal chicken anti-vimentin antibody	abcam	ab24525
Biotin-SP-conjugated, Donkey Anti-Mouse IgG	Jackson ImmunoResearch	715-065-150
StreptAvidin, Alex Flour 488 conjugated	Invitrogen	S32354
Rhodamine-conjugated Donkey Anti-Goat IgG	Jackson ImmunoResearch	705-025-147
Rhodamine-conjugated Donkey Anti-Guinea Pig IgG	Jackson ImmunoResearch	706-025-148
Rhodamine-conjugated Donkey Anti-Rabbit IgG	Jackson ImmunoResearch	711-025-152
Cy 5-conjugated Donkey Anti-Guinea Pig IgG	Jackson ImmunoResearch	706-175-148
Cy 5-conjugated Donkey Anti-Goat IgG	Jackson ImmunoResearch	705-175-147
Cy 5-conjugated Donkey Anti-Rabbit IgG	Jackson ImmunoResearch	711-175-152
Cy 5-conjugated Donkey Anti-Chicken IgG	Jackson ImmunoResearch	703-175-155
DAPI	Millipore	S7113
Disposable Reagent Reservoir 25 ml	Sorenson BioScience	39900
384 well, black/clear, tissue culture treated plate	BD Falcon	353962
96 well, black/clear, tissue culture treated plate	Costar	3603
Multi-channel pipettor	Costar	4880
12-channel vaccume aspirator	Drummond	3-000-096
Cell Scraper	Falcon	353085
Isotemp Water Bath Model 2223	Fisher Scientific
High-content screening instrument: ArrayScan VTI	Thermo Scientific