Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma

Summary

Intensive preparation of intact mouse cerebral endothelial "tubes" from cerebral parenchymal arterioles is illustrated for studying cerebral blood flow regulation. Further, we demonstrate the experimental strengths of this endothelial study model for fluorescence imaging and electrophysiology measurement of key cellular signaling pathways, including changes in intracellular [Ca²⁺] and membrane potential.

Abstract

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow increases with decreasing diameter from arteries to arterioles that ultimately feed into capillaries. Due to their smaller size and location in the parenchyma, arterioles have been relatively understudied and with less reproducibility in findings than surface pial arteries. Regardless, arteriolar endothelial cell structure and function—integral to the physiology and etiology of chronic degenerative diseases—requires extensive investigation. In particular, emerging evidence demonstrates that compromised endothelial function precedes and exacerbates cognitive impairment and dementia.

In the parenchymal microcirculation, endothelial K⁺ channel function is the most robust stimulus to finely control the spread of vasodilation to promote increases in blood flow to areas of neuronal activity. This paper illustrates a refined method for freshly isolating intact and electrically coupled endothelial "tubes" (diameter, ~25 µm) from mouse brain parenchymal arterioles. Arteriolar endothelial tubes are secured during physiological conditions (37 °C, pH 7.4) to resolve experimental variables that encompass K⁺ channel function and their regulation, including intracellular Ca²⁺ dynamics, changes in membrane potential, and membrane lipid regulation. A distinct technical advantage versus arterial endothelium is the enhanced morphological resolution of cell and organelle (e.g., mitochondria) dimensions, which expands the usefulness of this technique. Healthy cerebral perfusion throughout life entails robust endothelial function in parenchymal arterioles, directly linking blood flow to the fueling of neuronal and glial activity throughout precise anatomical regions of the brain. Thus, it is expected that this method will significantly advance the general knowledge of vascular physiology and neuroscience concerning the healthy and diseased brain.

Introduction

Parenchymal arterioles directly deliver essential oxygen and nutrients throughout the brain¹. While interfacing with capillaries, highly vasoactive arterioles respond to retrograde signaling initiated by capillary ion channels that sense metabolic signals from specific neuronal regions². With brain parenchyma having historically received the bulk of investigation, a role for endothelial dysfunction has now emerged for clarifying pathological mechanisms associated with various cerebrovascular disorders that underlie dementia (e.g., ischemic stroke, Alzheimer's disease)^3,4,5,6. The endothelium is integral to perfusion of the brain in accord with the heterogeneity of genetics, structure, and function throughout vascular segments⁷. Pial arteries have been extensively studied due to their relatively large size, high segmental vascular resistance, and role in blood flow distribution to the underlying cerebrum^8,9. Thus, a better understanding of arteriolar endothelial mechanisms will likely enhance the understanding of brain blood flow regulation in health and disease towards the development of novel therapeutic regimens.

Emerging evidence highlights the importance of studying parenchymal arterioles in relation to different signaling pathways and diseases^8,10. However, this approach has been limited to using intact pressurized arteriole¹¹ and/or capillary-parenchymal arteriole (CaPA) preparations¹². Freshly isolated, native cerebral arteriolar endothelial cells devoid of other cell types and confounding factors have not been examined, likely due to technical difficulties in their isolation. This paper advances a previous technique highlighting the isolation of pial arterial endothelium¹³ to now reliably and reproducibly isolate the endothelium of brain parenchymal arterioles (width: ~25 µm, length: ~250 µm). This technique helps achieve optimal resolution of electrically and chemically coupled cells in their individual orientation and cellular networks.

Key pathways of interest have included the interaction of intracellular Ca²⁺ ([Ca²⁺]_i) signaling and hyperpolarization of membrane potential (V_m)^14,15—integral to vasodilation¹⁶—to allow blood to enter the capillaries and deliver oxygen and nutrients to active parenchyma¹⁷. These preparations allow for real-time electrophysiological recordings of ion channels, including Ca²⁺-permeant, transient receptor potential (TRP) and K⁺ channels and/or fluorescent imaging of intracellular organelles within endothelial cell tubes in near-physiological conditions. This is a suitable technique for researchers interested in physiological cellular mechanisms that govern endothelial cell control of cerebral blood flow delivery to the brain parenchyma. Altogether, this technique will help researchers better understand fundamental endothelial signaling pathways and network communication of arterioles embedded in brain parenchyma while addressing questions related to cerebrovascular physiology and pathology.

Protocol

Experimenters should ensure that designated use of animals and associated protocols are approved by their Institutional Animal Care and Use Committee (IACUC) and performed in accordance with the National Research Council's "Guide for the Care and Use of Laboratory Animals" (8^th Edition, 2011) and the ARRIVE guidelines. The IACUC of Loma Linda University and the University of Arizona has approved all protocols used for this manuscript for C57BL/6N and 3xTg-AD mice (males and females; age range: 2-30 months). See Figure 1 as an overview of the isolation and examination of arteriolar endothelial tubes freshly isolated from mouse parenchymal arterioles of the brain.

1. Materials and equipment

NOTE: See the Table of Materials for all reagents and materials required for this protocol. In addition, manuals and websites associated with the respective vendors can also be consulted as needed. Illustrations of dissection stations and experimental apparatuses have been previously provided¹³.

1. Flow chamber
   1. Fasten a superfusion chamber with a glass coverslip onto a platform composed of anodized aluminum. Secure the platform with the chamber onto an aluminum microscope stage.
   2. Set a micromanipulator holding a pinning pipette at each end of the platform on the aluminum microscope stage.
      NOTE: If necessary, use a transferable stage apparatus with a flow chamber unit to move a secured, isolated endothelial tube from one microscope apparatus to another for experimentation.
2. Microscopes
   1. Use stereomicroscopes (5x to 50x magnification range) and fiber optic light sources for dissection procedures.
   2. For isolation of endothelial tubes, use an inverted microscope rig equipped with phase contrast- or differential interference contrast (DIC)-compatible objectives (10x, 20x, and 40x) and an aluminum stage.
   3. Have a microsyringe pump controller ready next to the apparatus dedicated to isolating endothelial tubes from partially digested blood vessels.
   4. Set up the experimental apparatus by arranging an inverted microscope (objectives: 4x, 10x, 20x, 40x, and 60x) and a manual aluminum stage on a vibration isolation table.
3. Intracellular V_m recording equipment
   1. Connect the electrometer to a compatible headstage. Use accessories, such as a function generator and stimulator, for protocols requiring current injection.
   2. Connect amplifier outputs to a data digitizer system, oscilloscope and audible baseline monitors. Secure the reference bath electrode (Ag/AgCl pellet) near the flow chamber exit.
   3. Assemble a photometric system with integrated components of a fluorescence system interface, high-intensity arc lamp and power supply, hyperswitch, photomultiplier tube (or PMT), and camera to measure [Ca²⁺]_i in endothelial cells.
   4. Assemble a temperature controller equipped with an inline heater to raise and maintain a physiological temperature (37 °C) throughout the experiment.
   5. Assemble a multichannel platform connected to a valve controller with an inline flow control valve to control delivery of solutions to endothelial tubes secured in the chamber.
4. Micropipettes and sharp electrodes
   ​NOTE: The experimenter will need an electronic glass puller and a microforge for preparing trituration and pinning pipettes.
   1. To separate the endothelial tube from the partially digested arteriolar segment, prepare heat-polished trituration pipettes (internal tip diameter: 30-50 µm) from borosilicate glass capillaries.
   2. To secure the endothelial tube in the superfusion chamber, prepare heat-polished pinning pipettes with a blunted, spherical end (outer diameter: 50-70 µm) prepared from thin-wall borosilicate glass capillaries.
   3. To record V_m of an endothelial cell, prepare sharp electrodes with a tip resistance of ~150 ± 30 MΩ from glass capillaries using the glass puller only.

2. Solutions and drugs

1. Physiological salt solution (PSS)
   1. Prepare a minimum of 1 L of PSS using 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 10 mM glucose.
   2. Prepare necessary solutions lacking CaCl₂ (zero Ca²⁺ PSS) for dissection of cerebral arterioles and isolation of endothelial tube.
      NOTE: Prepare all solutions in ultrapure deionized H₂O, followed by filtration (0.22 µm). Ensure that the final product contains a pH 7.4 with osmolality between 290 and 300 mOsm.
2. Bovine serum albumin (BSA, 10%)
   1. Dissolve 1 g of the lyophilized BSA powder in a beaker of 10 mL of zero Ca²⁺ PSS. Cover the beaker and allow at least 2 h with slow stirring for the BSA to dissolve.
   2. Filter solution using a 10 mL syringe plus a 0.22 µm filter and prepare 1 mL aliquots to be stored at -20 °C.
3. Dissection solution
   1. Add 500 µL of BSA (10%) to 49.5 mL of zero Ca²⁺ PSS for a total volume of 50 mL.
   2. Transfer solution to a Petri dish for arteriolar dissections.
4. Dissociation solution
   1. Add 5 µL of CaCl₂ solution (1 M) and 500 µL of BSA (10%) to 49.5 mL of zero Ca²⁺ PSS for a total volume of 50 mL. Incubate solution at room temperature for at least 1 h.
5. Enzymes
   1. Prepare 1 mL of digestion solution with 100 µg/mL papain, 170 µg/mL dithioerythritol, 250 µg/mL collagenase (Type H blend), and 40 µg/mL elastase.
      NOTE: Enzyme activities can vary, although successful protocols will likely require the following enzymatic activities: ≥ 10 units/mg for papain, ≥ 1 unit/mg for collagenase, and ≥ 5 units/mg for elastase.
6. Fura-2 and pharmacological agents
   1. Prepare Fura-2 AM stock in dimethyl sulfoxide (DMSO; 1 mM). Prepare 500 µL of working concentration (10 µM) by adding 5 µL of the stock to 495 µL of PSS for loading.
   2. Prepare at least 50 mL of working concentrations of pharmacological agents in PSS or DMSO as appropriate.
7. Conducting solution
   1. Prepare 2 M KCl by dissolving KCl in deionized H₂O (7.455 g of KCl in 50 mL of H₂O). Pass the solution through a syringe with a 0.22 µm filter prior to backfilling the sharp electrodes.
8. Fluorescent organelle trackers and antibodies
   1. Prepare plasma membrane or organellar (e.g., nucleus, endoplasmic reticulum) trackers in the appropriate physiological saline solution according to the manufacturer's instructions.
   2. Prepare primary and secondary antibodies according to the manufacturer's instructions in the appropriate physiological salt solution.

3. Dissection and isolation of mouse cerebral arterioles

NOTE: Stereomicroscopes and sharpened microdissection tools (e.g., fine-tipped forceps, Vannas-style dissection scissors) must be used for specimen magnification (up to 50x) in all these dissection procedures.

1. Isolation of mouse brain
   1. Anesthetize a standard laboratory mouse (e.g., C57BL/6, 3xTg-AD; 2-30 months old, male or female) using isoflurane inhalation (3% for 2-3 min), followed by immediate decapitation using sharp scissors or guillotine per IACUC approval. Place the mouse head in a Petri dish (diameter 10 cm, depth 1.5 cm) containing cold (4 °C) dissection solution.
   2. While viewing under a stereomicroscope, remove the skin and hair over the skull and wash away excessive blood with cold dissection solution. Using the tips of standard dissection scissors (e.g., 24 mm blades), make an incision starting with the occipital bone and extending up through the nasal bone of the skull. Use coarse-tipped forceps to carefully open the skull along the incision, and separate connective tissue (meningeal membrane) to isolate the brain containing an intact Circle of Willis.
   3. Wash the isolated brain with cold dissection solution in a beaker or Petri dish to remove remaining blood from the surface of the brain. Place the brain ventral side facing up in a chamber containing cold dissection solution for isolation of parenchymal arterioles (Figure 2A).
2. Isolation of parenchymal arterioles
   NOTE: Arterioles arising from the middle cerebral arteries (MCAs) and embedded in brain parenchyma are chosen for this protocol. The arterioles are isolated as previously described¹¹, with modification.
   1. Use steel pins (diameter: 0.2 mm, length: 11 to 12 mm) to secure the isolated brain in cold dissection solution in a Petri dish containing a charcoal-infused silicon polymer coating (depth ≥ 50 cm).
   2. Using sharp and aligned dissection scissors, cut a rectangle of brain tissue (length: 5 mm, width: 3 mm) (Figure 2B) around the MCA while ensuring that the upper part of the tissue segment is past the branching point from the Circle of Willis. Cut another rectangle of brain tissue from the other hemisphere of the brain to access more arterioles if necessary.
   3. Secure the separated brain tissue segment into the dish with the MCA facing upwards (distal from the Circle of Willis) using steel pins (diameter: 0.1 mm, length: 13 to 14 mm). Carefully make a shallow incision near the pins to remove the pia with small forceps, gently peeling from one end towards the other (Figure 2C). Remove the pia from the other tissue segment to access more arterioles if needed. Carefully secure the isolated pia with parenchymal arterioles branched from MCA in the dish with the pins, and dissect the parenchymal arterioles (Figure 2D), ensuring that the arterioles are not damaged.
      NOTE: If the arterioles are not easily pulled out with the pia from the parenchyma, use fine forceps and dig into the brain, starting at the MCA branch point from the Circle of Willis. Locate the arteriole, carefully loosen the tissue around arterioles (Figure 2E), and gently pull the arteriole out of the parenchyma, holding the upper end of the arteriole (Figure 2F). Multiple arterioles (length: ~1.5-2 mm) can be isolated from one rectangle of brain tissue.
   4. Ensure the arteriole is clean with no tissue attached to it, and cut off any remaining distal branches (Figure 3A). Use this clean, intact arteriole for enzymatic digestion. Alternatively, cut each arteriole into two pieces (length: 0.75-1 mm) for enzymatic digestion for the preparation of endothelial tubes if desired.

4. Digestion of parenchymal arterioles and preparation of endothelial tubes

1. Arrangement of equipment and pipettes
   NOTE: Isolation of arterial endothelial tubes from the brain has been described previously^13,18. The current protocol incorporates modifications for the isolation of endothelium from parenchymal (intracerebral) arterioles. Multiple arterioles or/and pieces of arterioles can be used together for enzymatic digestion.
   1. Assemble the trituration apparatus¹³ for preparing endothelial tubes using an aluminum stage supporting a chamber and micromanipulators. Assemble a microscope equipped with objectives (5x-60x) and a camera connected to a computer monitor. Secure a microsyringe with a pump controller next to the stage and specimen.
   2. Secure a trituration pipette backfilled with mineral oil over the microsyringe piston. Use the microsyringe with the pump controller to withdraw the dissociation solution into the trituration pipette (~130 nL) on top of the mineral oil while taking care to avoid air bubbles in the pipette.
2. Partial digestion of arteriolar segments
   1. Place intact arteriolar segments into 1 mL of dissociation solution in a 10 mL glass tube containing 100 µg/mL papain, 170 µg/mL dithioerythritol, 250 µg/mL collagenase, and 40 µg/mL elastase. Incubate arteriolar segments at 34 °C for 10-12 min.
   2. After digestion, replace the enzyme solution with 3-4 mL of fresh dissociation solution at room temperature.
3. Isolation of arteriolar endothelial tube
   NOTE: Following digestion and replacement of the enzyme solution, transfer one or multiple partially digested segments into the dissociation solution in a superfusion chamber attached to a mobile platform. While viewing at 100x to 200x magnification, select one partially digested but unbroken vessel segment and focus on it under the microscope.
   1. Place the trituration pipette attached with the microsyringe injector in the dissociation solution in the chamber and position it close to one end of the digested vessel segment. Set a rate within the range of 1-3 nL/s on the pump controller for gentle trituration.
   2. While maintaining 100x to 200x magnification, withdraw the arteriolar segment into the pipette and then eject to dissociate the adventitia and smooth muscle cells (Figure 3B). Triturate the vessel segment until all smooth muscle cells are dissociated, and only endothelial cells remain as an intact "tube."
      NOTE: If necessary during trituration, carefully use fine-tipped forceps to remove the dissociated adventitia and internal elastic lamina from the endothelial tube. Typically, only a few cycles of trituration yield an intact endothelial tube.
   3. Use micromanipulators to secure each end of the endothelial tube on the glass coverslip in the chamber with borosilicate glass pinning pipettes (Figure 3C,D).
4. Securing the arteriolar endothelial tube
   1. Replace the dissociation solution with superfusion solution (PSS containing 2 mM CaCl₂) while ensuring that nonendothelial material is washed out of the chamber.
   2. Transfer secured endothelial tube in the mobile platform to the microscope rig designed for continuous superfusion and intracellular or fluorescent recordings/imaging.
   3. Apply continuous delivery of PSS during the experiment. Manually set the flow rate (5-7 mL/min) throughout the experiment using the inline flow control valve, consistent with the laminar flow, while matching the feed of solution flow to the extent of vacuum suction. Allow ≥5 min of PSS flow to the chamber for superfusion of the endothelial tube before acquiring background data and/or dye loading.

5. Utilization of arteriolar endothelial tubes for the examination of cellular physiology

NOTE: Isolated and secured arteriolar endothelial tubes can be used for intracellular recordings of [Ca²⁺]_i dynamics and V_m using photometry and sharp electrode electrophysiology, respectively, as previously illustrated¹³ (Figure 4). [Ca²⁺]_i and V_m can be measured as separate or combined experimental variables as needed¹³ (Figure 4). However, arteriolar endothelial tubes are more delicate than arterial endothelium, and experimentation time should not exceed 1 h.

1. Measurement of [Ca²⁺]_i
   1. Turn on the equipment and software for [Ca²⁺]_i recordings while maintaining continuous superfusion at a flow rate of 5-7 mL/min.
   2. Load the endothelial tube with the Ca²⁺ dye Fura-2 AM for 30 min at room temperature. Wash the cells with superfusion solution for another 20-30 min while gradually raising the bath temperature to 37 °C. Maintain the temperature at 37 °C throughout the experiment.
   3. Manually adjust the imaging window using photometry software to focus on ~20 endothelial cells (Figure 4A). In the absence of light, turn the PMT on the fluorescence interface and begin acquisition of [Ca²⁺]_i by exciting Fura-2 alternately (≥10 Hz) at 340 nm and 380 nm while collecting fluorescence emission at 510 nm. Once a stable baseline recording of [Ca²⁺]_i is established, apply pharmacological agents (e.g., purinergic receptor agonists) per experimental objective (Figure 4B).
2. Measurement of V_m​
   1. Turn on the equipment and software for V_m recordings and set the data acquisition rate (≥10 Hz) while maintaining continuous superfusion at a flow rate of 5-7 mL/min. Gradually raise the bath temperature to 37 °C and maintain it until the end of the experiment.
   2. Pull a sharp electrode using a borosilicate glass capillary, backfill with 2 M KCl, and secure it over a silver wire coated with chloride in the pipette holder attached to an electrometer, that in turn, is held by a micromanipulator.
   3. While viewing through the 4x objective, use a micromanipulator to carefully position the sharp electrode tip just over a cell of the arteriolar endothelial tube into the flowing PSS in the chamber.
   4. Gradually increase magnification to 400x and reposition the electrode tip as needed.
   5. Using the micromanipulator, gently insert the tip of a sharp electrode into one of the cells of the endothelial tube and start recording V_m using an electrometer (Figure 4A).
   6. Once the endothelial resting V_m is stable (−30 to −40 mV), apply the desired pharmacological agents per experimental objective (Figure 4C).

6. Cellular imaging

NOTE: Endothelial tubes secured in the chamber of the mobile platform can also be used for microscopic imaging in both live and fixed conditions¹⁹ using standard fluorescence or confocal microscopy. Immunohistochemistry with different antibodies for receptors and ion channels can also be applied as previously described²⁰.

1. Load the endothelial tube with the fluorescence tracker for the plasma membrane or desired organelle (e.g., nucleus, endoplasmic reticulum) at 37 °C for 15-30 min.
2. Wash the cells with fresh superfusion PSS and image live cells under the microscope at the excitation wavelength of respective dyes (Figure 4D,E).
   NOTE: Immunohistochemistry can be performed on this tube model using antibodies of interest.

Results

A demonstration of the protocol is shown in Figure 1 with arteriolar dissection and endothelial tube isolation steps as Figure 2 and Figure 3, respectively. Here, endothelial function was assessed by measuring [Ca²⁺]_i and V_m using Fura-2 photometry and sharp electrode electrophysiology (Figure 4A) in response to a pharmacological agent [2-methylthioadenosine diphosphate ...

Discussion

Growing evidence suggests that cerebrovascular disease (CVD), aging, and Alzheimer's disease are strongly correlated and are a current topic of dementia research^4,8,14,21. Thus, it is obvious that studies of the cerebrovascular network would have a broad impact on health while requiring continued extensive investigation during conditions of disease. As a significant point of vascular r...

Materials

Name	Company	Catalog Number	Comments
Amplifiers	Molecular Devices, Sunnyvale, CA, USA	Axoclamp 2B & Axoclamp 900A
Audible baseline monitors	Ampol US LLC, Sarasota, FL, USA	BM-A-TM
Bath Chiller (Isotemp 500LCU)	ThermoFisher Scientific	13874647
Borosilicate glass capillaries (Pinning)	Warner Instruments	G150T-6
Borosilicate glass capillaries (Sharp Electrodes)	Warner Instruments	GC100F-10
Borosilicate glass capillaries (Trituration)	World Precision Instruments (WPI), Sarasota, FL, USA	1B100-4
BSA: Bovine Serum Albumin	Sigma	A7906
CaCl2: Calcium Chloride	Sigma	223506
Collagenase (Type H Blend)	Sigma	C8051
Cover Glass (2.4 × 5.0 cm)	ThermoFisher Scientific	12-548-5M
Data Acquision Digitizer	Molecular Devices, Sunnyvale, CA, USA	Digidata 1550A
Dissection Dish (Glass Petri with Charcoal Sylgard bottom)	Living Systems Instrumentation, St. Albans City, VT, USA	DD-90-S-BLK
Dithioerythritol	Sigma	D8255
DMSO: Dimethyl Sulfoxide	Sigma	D8418
Elastase (porcine pancreas)	Sigma	E7885
Endoplasmic Reticulum Tracker (ER-Tracker Red, BODIPY TR Glibenclamide)	ThermoFisher Scientific	E34250
Fiber optic light sources	Schott, Mainz, Germany & KL200, Zeiss	Fostec 8375
Flow Control Valve	Warner Instruments	FR-50
Fluorescence system interface, ARC lamp & power supply, hyperswitch and PMT	Molecular Devices, Sunnyvale, CA, USA	IonOptix Systems
Forceps (Fine-tipped, sharpened)	FST	Dumont #5 & Dumont #55
Function Generator	EZ Digital, Seoul, South Korea	FG-8002
Fura-2 AM dye	Invitrogen, Carlsbad, CA, USA	F14185
Glucose	Sigma-Aldrich (St. Louis, MO, USA)	G7021
HCl: Hydrochloric Acid	ThermoFisher Scientific (Pittsburgh, PA, USA)	A466250
Headstages	Molecular Devices	HS-2A & HS-9A
HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)	Sigma	H4034
Inline Solution Heater	Warner Instruments	SH-27B
KCl: Potassium Chloride	Sigma	P9541
MgCl2: Magnesium Chloride	Sigma	M2670
Microforge	Narishige, East Meadow, NY, USA	MF-900
Micromanipulator	Siskiyou	MX10
Micropipette puller (digital)	Sutter Instruments, Novato, CA, USA	P-97 or P-1000
Microscope (Nikon-inverted)	Nikon Instruments Inc, Melville, NY, USA	Ts2
Microscope (Nikon-inverted)	Nikon Instruments Inc	Eclipse TS100
Microscope objectives	Nikon Instruments Inc	20X (S-Fluor) and 40X (Plan Fluor)
Microscope platform (anodized aluminum; diameter, 7.8 cm)	Warner Instruments	PM6 or PH6
Microscope Stage (Aluminum)	Siskiyou, Grants Pass, OR, USA	8090P
Microsyringe Pump Controller	World Precision Instruments (WPI), Sarasota, FL, USA	SYS-MICRO4
MTA: 2-Methylthioadenosine diphosphate trisodium salt	Tocris	1624
NaCl: Sodium Chloride	Sigma	S7653
NaOH: Sodium Hydroxide	Sigma	S8045
Nuclear Stain (NucBlue Live ReadyProbes Reagent; Hoechst 33342)	ThermoFisher Scientific	R37605
Oscilloscope	Tektronix, Beaverton, Oregon, USA	TDS 2024B
Papain	Sigma	P4762
Phase contrast objectives	Nikon Instruments Inc	(Ph1 DL; 10X & 20X)
Plasma Membrane Stain (CellMask Deep Red)	ThermoFisher Scientific	C10046
Plexiglas superfusion chamber	Warner Instruments, Camden, CT, USA	RC-27
Scissors (3 mm & 7 mm blades)	Fine Science Tools (or FST), Foster City, CA, USA	Moria MC52 & 15000-00
Scissors (Vannas style; 9.5 mm & 3 mm blades)	World Precision Instruments	555640S, 14364
Stereomicroscopes	Zeiss, NY, USA	Stemi 2000 & 2000-C
Syringe filter (0.22 µm)	ThermoFisher Scientific	722-2520
Temperature Controller (Dual Channel)	Warner Instruments	TC-344B or C
Valve Control System	Warner Instruments	VC-6
Vibration Isolation Table	Technical Manufacturing, Peabody, MA, USA	Micro-g