A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Summary

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

Abstract

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert's membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

Introduction

The tumor microenvironment (TME) is a highly complex system comprised of carcinoma cells that co-exist and evolve alongside host stroma. This stromal component typically consists of fibroblasts, myofibroblasts, endothelial cells, various immune components, as well as an extracellular matrix¹. A significant constituent, often the majority of this stroma, are activated fibroblasts, frequently referred to as cancer-associated fibroblasts or carcinoma-associated fibroblasts (CAF)^2,3. Unlike normal, non-activated fibroblasts, CAFs contribute to tumor initiation, progression, angiogenesis, invasion, metastasis, and recurrence^4-11 in a wide variety of carcinomas, including breast, prostate, lung, pancreas, skin, colon, esophagus, and ovary^5,6,12-17. Yet, the exact nature of the contribution of CAFs throughout cancer pathogenesis remains poorly defined. Furthermore, clinical evidence has demonstrated a prognostic value of CAFs, correlating their presence to high-grade malignancies, therapy failure, and overall poor prognosis^10,18,19.

Clearly, enhancing our understanding of the initiating events in CAF development, as well as the intercellular communications mediating their role within the TME, may provide exciting new therapeutic targets and enhanced strategies that could improve patient outcomes. Towards this goal, several in vivo and in vitro models have been developed. While in vivo approaches are more reflective of patients' TME, they possess limitations, including the immense complexity and heterogeneity both within and between tumors. Furthermore, tumor samples from human subjects often represent highly developed TME and do not permit an understanding of the TME initiating events. Experimental animal studies offer some advantages, however generalization of animal data to humans should be done with caution due to differences in physiology between humans and animals such as rodents (e.g., thiol chemistry²⁰, metabolic rate²¹, tolerance to stress²², etc.). Further, unlike the human population, which is genetically heterogeneous in nature, laboratory animals are typically bred to homogeneity. Also, it is often difficult to examine transient physiological variations and cell phenotype changes, as well as to control for specific experimental parameters using animals such as rodents. Thus, in vitro 2- and 3-dimensional (2D and 3D) tissue culture models are frequently utilized to advance the basic understanding of TME development. In spite of their lack of an accurate portrayal of the complexity of in vivo systems, these models offer advantages that greatly facilitate mechanistic investigations. In vitro models allow for a more simplified, focused, and cost-effective analysis of the TME, whereby statistically significant data can be generated in cells free of systemic variations that arise in animals.

There are several varieties of in vitro systems. The two most commonly used TME in vitro models consist of mixed monolayer or spheroid cell cultures. Both culture methods are advantageous for basic studies of intercellular interactions (e.g., normal cells with tumor cells) and for the analysis of various TME specific cell phenotype changes (e.g., emergence of cancer-associated fibroblasts from normal fibroblasts). Additionally, the spheroids are able to create a more reflective tissue-like structure of the TME, and can be representative of tumor heterogeneity²³. However spheroids often produce widely varying oxygen tension gradients across layers, which may complicate experimental conclusions²⁴. Unfortunately, both models are extremely limited in their ability to isolate pure cell populations for further characterization and study following co-culture. To do so would require at least one cell type to be fluorescently-tagged or labeled with an identifying maker, and then subjecting the mixed co-culture to extensive processing and cell sorting to separate the cell populations. While a cell sorter is capable of isolating a rather pure cell population, one must be cognizant of cellular stress and potential microbial contamination risks²⁵.

To facilitate the understanding of intercellular communication, great efforts have been devoted towards developing and optimizing in vitro systems that closely mimic the in vivo environment, while permitting a simplified approach. One such tool is the permeable microporous insert, a membrane substrate that was first developed in 1953²⁶ and subsequently adapted for diverse applications and studies (e.g., cell polarity²⁷, endocytosis²⁸, drug transport²⁹, tissue modeling³⁰, fertilization³¹, bystander effect^32,33, etc.). This system permits the growth of cells with in vivo-like anatomical and functional differentiation, as well as expression of many in vivo markers^34,35 that are not observed when cultured on impermeable plasticware. Furthermore, the extremely thin porous membrane (10 µm thick) permits rapid diffusion of molecules and equilibration times, which simulates the in vivo environment and permits independent cellular functioning at both the apical and basolateral cell domains. An additional advantage of the insert's utility as a TME system is its physical separation of two heterotypic cell populations grown on either side of the membrane in the same environmental conditions, while maintaining various modes of intercellular communication through the membrane pores. Though physically separated, the two cell populations are metabolically coupled via secreted elements and, as described here, also through gap-junctional channels. Additionally, by maintaining the inserts at in vivo partial oxygen tension (PO₂), the model reduces the complications of oxygen and chemical gradients observed in other systems. Rather, it increases the understanding of natural mechanisms controlling the TME. Notably, the two cell populations can be easily isolated with high purity, without fluorescent tagging and/or cell sorting following extended periods of co-culture.

Here we describe an in vitro TME protocol consisting of human breast carcinoma cells and human fibroblasts grown, respectively, on either side of a permeable microporous membrane insert, but yet in continuous bi-directional communication through the membrane pores. We show that by using membranes with different pore sizes, the contribution of a specific type of intercellular communication (e.g., secreted factors versus gap junctions) to the development of the TME can be investigated.

Protocol

1. Preparation of Culture Media and Cells

1. Prepare 500 ml of Eagle's minimum essential medium supplemented with 12.5% (vol/vol) heat-inactivated fetal bovine serum (FBS), 2 mM L-alanyl-L-glutamine, and 100 units of penicillin and 100 µg of streptomycin per ml.
   NOTE: The growth medium and supplement(s) can be easily exchanged for the growth requirements of other cell strains or cell lines.
2. Prepare 70 µl of cell culture medium for each insert (6-well format insert): Eagle's minimum essential medium supplemented with 50% (vol/vol) heat-inactivated FBS, 2 mM L-alanyl-L-glutamine, and 100 units of penicillin and 100 µg of streptomycin per ml.
   NOTE: The medium is supplemented with 50% FBS to facilitate cell attachment to the bottom side (i.e., underside) of the insert. The growth medium and supplement(s) can be easily exchanged for the growth requirements of other cell strains or cell lines.
3. Prepare a cell suspension of the cells destined to be plated on the bottom side of the insert.
   NOTE: Here, the results were obtained using AG1522 normal human fibroblasts grown in 75 cm² cell culture flasks, and these cells will therefore be the focus of description of the cell suspension's preparation.
4. To collect cells, remove the growth medium and wash the cell monolayer 2x with 5 ml 1x Phosphate Buffered Saline (PBS).
5. Remove the PBS and spread 1 ml of 0.25% (vol/vol) trypsin with 2.21 mM ethylenediaminetetraacetic acid (EDTA), pre-warmed to room temperature (RT) over the cells for 2 min at RT (room temperature).
6. Quench the trypsin activity with 9 ml of complete growth medium (prepared in step 1.1) and collect cells by gently pipetting the cell suspension over the surface of the flask 10x.
7. Determine the cell concentration by using a hemocytometer or electronic counter and pipette the cell suspension volume that will provide 250,000 cells per insert into a sterile 15 ml centrifuge tube.
8. Pellet the cell suspension by centrifugation (800 x g, 2 min). Suspend the cell pellet with growth medium (prepared in step 1.2) to create a concentration of 250,000 cells per 70 μl.
   NOTE: As cell culture in permeable microporous membrane inserts is based on 2D substrates, cell seeding is performed similarly, except the cells should be initially suspended in medium containing 50% (vol/vol) FBS to facilitate attachment.

2. Preparation of Inserts

NOTE: To ensure sterile conditions, work in a laminar flow biological safety cabinet dedicated to cell culture.

1. Select inserts with a membrane pore size of 0.4 μm, 1 μm, or 3 μm (determined by the experimental interest(s), as the pore size can be used to explore the role of different modes of intercellular communication).
   NOTE: The 0.4 μm pore is small enough to limit formation of functional gap junctions between cells on either side of the insert membrane and can be used to study intercellular communication by secreted factors. Whereas, the 1 μm and 3 μm pores are large enough to allow formation of functional gap junctions and can be used to study intercellular communication by both gap junctions and secreted factors.
2. Remove individual inserts from the packaging and place into an equally sized multi-well dish.
   NOTE: Inserts are available with various membrane surface areas to fit specific application needs (e.g., 6-well, 12-well, and 24-well inserts). Here, the results were obtained using the 6-well insert, and will therefore be the focus of the model description.
3. Cover the dish, containing the inserts with the dish lid. Holding the multi-well dish with both hands, gently invert the dish such that the insert bottoms (i.e., underside) are now facing upwards.
4. Remove the bottom of the multi-well dish, exposing the bottom side of the inserts. Working with one insert at a time, use a sterile pair of forceps and gently hold the insert in place. With the free hand, use a pipette with a wide-mouth tip to draw 70 μl of the cell suspension (prepared in steps 1.3-1.8), containing 250,000 cells.
5. To plate the cells, place the pipette tip gently on the center of the insert's membrane, and slowly pipette the 70 μl of cell suspension while slowly moving the pipette tip around the surface of the membrane. Be careful not to extend the pipette tip and cell suspension to the edge of the insert.
   NOTE: Touching the edge of the insert could result in loss of capillary tension of the cell suspension leading to the cells drying out during attachment.
6. Repeat for remaining inserts, with care being exercised not to work directly over the exposed inserts to avoid potential microbial contamination.
7. Gently return the multi-well dish bottom to cover the inserts. Keeping the dish with inserts inverted, incubate in a humidified air atmosphere of 5% (vol/vol) CO₂ at 37 °C for 30-45 min.
   NOTE: If the cell suspension on the insert contacts the bottom of the well, carefully lift the dish bottom and rest it on the edge of the dish top, so that it forms a slight angle and creates greater separation between the surface of the dish and the bottom side of the inserts (where the cells are seeded). This will prevent the cells from attaching to the surface of the wells instead of the insert.
8. Following the 30-45 min incubation time, gently remove the dish containing the inserts and place it in the laminar flow biological safety cabinet.
9. With two hands and keeping the dish lid tight, gently re-invert the dish such that the bottom side of the inserts containing the attached cells now faces down.
10. Slowly and carefully add 2 ml (1 ml for 3 μm pore inserts to avoid migration of cells through the insert pores) of pre-warmed complete medium (see step 1.1) to each well, so that the bottom of the insert is immersed.
11. After adding growth medium in all the wells containing inserts, place the dish back into the humidified incubator.
12. Allow the inserts to remain undisturbed in the incubator for 48 hr. After 48 hr, feed the cells by aspirating the 2 ml of medium from the bottom of the well and adding 2 ml of fresh growth medium.
13. After adding fresh medium to the bottom of the well, seed 1 ml of the second cell population suspension (~250,000 cells/ml) on the top side of the insert, which already has the first cell population growing on the bottom side (for these experiments, AG1522 (control) cells or cancer (experimental) cells are plated on the top of the inserts, which already have AG1522 cells, destined to become CAFs, growing on the bottom side of the inserts). Place the dish back into the humidified incubator.
    NOTE: If working with cells that do not adhere well to the bottom of the insert, these cells can be alternatively grown on the top side of the insert.
14. Change the medium at 24, 72, and 96 hr after seeding the second population of cells on the top side of the insert by aspirating the medium from the top of the insert and bottom of the well. Gently add 1 ml of fresh medium to the top side of the insert and 2 ml to the bottom of the well. Allow the two cell populations to remain in co-culture on either side of the permeable microporous insert membrane for 120 hr.

3. Collecting Cells from Insert by Trypsinization

NOTE: In addition to the ease of obtaining enriched cell populations, the insert TME model also allows for similar experimental treatments (e.g., incubation with chemicals, exposure to oxygen conditions that are above or below ambient atmosphere, ionizing radiation treatment, etc.) as other in vitro TME co-culture models. Furthermore, the permeable insert co-culture substrate can be analyzed by procedures already developed for standard 2D tissue culture models. For example, cells can be harvested from either side of the insert membrane to obtain highly enriched populations, which can then be utilized for analysis of endpoints (e.g., in situ immuno-detection, Western blotting) or propagated for subsequent experiments³⁶.

1. To collect cells, remove the insert from the growth medium and place it into a 35 mm cell culture dish containing 1 ml PBS. Wash the bottom and top sides of the insert 1x with 1 ml PBS.
2. Remove the PBS and add 200 μl of 0.25% (vol/vol) trypsin with 2.21 mM EDTA, pre-warmed to RT.
   1. If collecting the cells grown on the bottom side of the insert, place the trypsin solution into the 35 mm dish and gently place the bottom of the insert into the trypsin solution for 2 min at RT.
   2. If collecting the cells grown on the top side of the insert, place the insert into a 35 mm dish and add the trypsin solution to the top of the insert and incubate for 2 min at RT.
3. Quench the trypsin activity by adding 800 μl of complete growth medium.
   1. If collecting the cells grown on the bottom of the insert, add the growth medium to the 35 mm dish and collect the cells by gently pipetting the 1 ml of cell suspension over the surface of the insert 10x, with the insert being held at a slight angle, such that the cell suspension collects into the dish.
   2. If collecting the cells from the top side of the insert, add the growth medium to the top side and gently pipette the 1 ml of cell suspension over the surface of the insert 10x.

4. Characterization of Cell Purity by Flow Cytometry

NOTE: To characterize the ability of the permeable microporous membrane inserts to maintain the purity of the two cell populations (i.e., top and bottom) for up to 120 hr, sections 1-3 were performed, with green fluorescent protein (GFP)-tagged MDA-MB-231 cells being plated on the top side of the insert, and non-GFP-tagged AG1522 cells being plated on the bottom side of 0.4 µm-, 1 µm-, or 3 µm pore inserts.

1. Once the cells from the bottom side of the insert were collected (procedure described in Section 3), pellet the cell suspension by centrifugation (500 g, 30 sec).
2. Remove the supernatant and suspend the cell pellet in 400 µl of Hanks' Balanced Salt Solution (HBSS) supplemented with 1% (vol/vol) FBS.
3. Perform cytometric analysis on a flow cytometer equipped with a 488 nm laser to excite GFP and a bandpass filter of 530/30 to detect GFP photon emission³⁷.
4. For cell identification purposes, use AG1522 control cell populations to adjust forward and side scatter voltages on the flow cytometer. Adjust the GFP channel voltage to set the cellular autofluorescence value as the lower detection threshold for the GFP signal.
5. Determine gating threshold using control cells. Assess purity of each cell population based upon the presence of GFP-positive cells versus control.
   NOTE: A pure population would be reflective of no GPF-positive cells detected.

5. Characterization of Cells by In Situ Immunofluorescence

1. Following trypsinization of cells from the insert (see section 3), collect cells, plate 5 x 10⁴ cells in 250 μl growth medium (see step 1.1) onto sterile glass coverslips, and allow to attach for 1 hr in a humidified air atmosphere of 5% (vol/vol) CO₂ at 37 °C incubator.
2. At the end of the incubation, gently remove the dish containing the coverslip and place it in the laminar flow biological safety cabinet.
3. Carefully add 2 ml of pre-warmed complete medium (see step 1.1) to each dish, so that the coverslip is immersed.
4. After adding growth medium to all the dishes containing coverslips, return the dishes to a humidified air atmosphere of 5% (vol/vol) CO₂ at 37 °C in an incubator for 48 hr.
5. Following the 48 hr incubation, wash the sample 3x with PBS and fix with 4% (wt/vol) formaldehyde in PBS for 10 min at RT.
6. Rinse 5x with Tris Buffered Saline (TBS: 50 mM Tris-Cl, pH 7.5, 150 mM NaCl) and then permeabilize with 0.25% (vol/vol) Triton X-100, 0.1% (wt/vol) saponin for 5 min at RT.
7. Block non-specific binding sites with 2% (vol/vol) normal goat serum (NGS), 1% (wt/vol) bovine serum albumin (BSA), 0.1% (vol/vol) Triton X-100 in TBS (blocking solution) for 1 hr at RT.
8. Incubate with primary antibody (anti-Caveolin 1 (CAV1), 1:1,000) in blocking solution at 4 °C overnight.
9. Wash off unbound primary antibodies (3x, 5 min/wash) with 0.2% NGS (vol/vol), 1% (wt/vol) BSA, 0.1% (vol/vol) Triton X-100 in TBS (wash solution).
10. Incubate with secondary antibody at RT for 1 hr in blocking solution (see section 5.4).
11. Wash off unbound secondary antibody (3x, 5 min/wash) with wash solution (see step 5.6).
12. Mount coverslips on a slide with an anti-fade mounting medium containing 4',6-diamidino-2-phenylindole (DAPI), and seal with nail polish.
13. Observe cells using a 63X oil magnification objective on an inverted microscope equipped with an external light source for fluorescence excitation, and capture images using an attached digital camera for subsequent analysis.

6. Characterization of Cells by Western Blot

NOTE: The Western blot procedure is described elsewhere³⁸. A brief outline is described here:

1. Following detachment of cells from the insert by trypsinization (see section 3), pellet the cell suspension by centrifugation (500 g, 30 sec).
2. Remove supernatant and suspend the cell pellet 2x with 100 µl PBS.
3. Remove supernatant and lyse cells in 50 µl of modified radioimmunoprecipitation assay (RIPA) buffer (Tris pH 7.5 50 mM, NaCl 150 mM, NP40 1% (vol/vol), sodium deoxycholate monohydrate (DOC) 0.5% (vol/vol), sodium dodecyl sulfate (SDS) 0.1% (vol/vol)), containing protease and phosphatase inhibitor cocktail.
4. Incubate for 20 min on ice and centrifuge at 19,000 g for 15 min at 4 °C.
5. Determine concentration of proteins in the supernatant using a protein optical density assay, compatible with the detergents in the RIPA buffer.
6. Separate proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblot onto a 0.2 µm nitrocellulose membrane.
7. Incubate membranes in TBS containing 0.1% (vol/vol) Tween-20 (TBST) and 4% (wt/vol) skim milk (blocking solution) for 60 min and react with primary antibody at 4 °C overnight (anti-CAV1, 1:1,000).
8. Wash membrane 3x with TBST at RT (5 min/wash).
9. Incubate membranes for 30 min in blocking solution containing an anti-mouse secondary antibody conjugated with horseradish peroxidase (HRP) (1:5,000).
10. Wash membrane 3x with TBST at RT (5 min/wash).
11. Visualize reaction products by chemiluminescence using a Western blotting detection system.

7. Characterization of Intercellular Communication by Flow Cytometry

NOTE: To characterize the adaptability of the permeable microporous membrane inserts to examine different modes of intercellular communication (e.g., secreted factors, gap junctions). Sections 1-2.12 were performed with non-fluorescent AG1522 cells being plated on the bottom side of 0.4 µm-, 1 µm-, or 3 µm-pore inserts.

1. Culture non-labeled AG1522 cells in a 35 mm dish to 90% confluency using medium described in section 1.1.
2. Rinse cells 1x with 1 ml of HBSS.
3. Load cells with Calcein AM by incubating cells in HBSS containing 30 µM of Calcein AM.
4. Incubate for 15 min in a humidified air atmosphere of 5% (vol/vol) CO₂ at 37 °C.
5. Wash cells 2x with 1 ml of HBSS.
6. Trypsinize cells by adding 200 µl solution of 0.25% (vol/vol) trypsin with 2.21 mM EDTA for 2 min at RT.
7. Quench the trypsin activity by adding 800 µl of complete medium containing 12.5% (vol/vol) FBS.
8. Bring cells into suspension by repeated pipetting.
9. Determine cell concentration by using a hemocytometer or electronic counter and plate 250,000 cells loaded with Calcein AM to the top side of inserts that already have AG1522 cells growing on the bottom side.
10. Incubate the inserts in a humidified air atmosphere of 5% CO₂ at 37 °C for 6 hr.
11. After 6 hr, collect cells from the bottom side of the inserts, as described in section 3.
12. Pellet the cell suspension by centrifugation (500 g, 30 sec).
13. Remove the supernatant and suspend the cell pellet in 400 µl of HBSS supplemented with 1% (vol/vol) FBS.
14. Analyze the cells on a cytometer equipped with a laser and filter capable of exciting and detecting the emission of Calcein (496 nm and 516 nm, respectively), per manufacturer protocol.
15. For cell identification purposes, use an unstained AG1522 control cell population to adjust forward and side scatter voltages on the flow cytometer. Adjust the Calcein channel voltage to set the cellular autofluorescence value as the lower detection threshold for the Calcein signal.
16. Determine the upper threshold limit using Calcein-loaded control cells. Assess communication for each cell insert based upon the presence of Calcein-positive cells versus control.

Results

Here we adapted a permeable microporous membrane insert to develop an in vitro heterotypic cell co-culture system that mimics the in vivo tumor microenvironment (Figure 1). This system allows for two different cell populations to be grown on either side of the insert's porous-membrane for extended periods of time (up to 120 hr, in our use). Importantly the system is capable of maintaining the purity of the cell populations, as determined by plating G...

Discussion

The protocol described here is a simple, adaptable in vitro procedure (Figure 1) that utilizes a permeable microporous membrane insert to generate highly enriched individual cell populations from a co-culture of heterotypic cells. Significantly, the model is suitable for investigating various modes of intercellular communication. The critical steps include selecting the appropriate pore-size insert for specific experimental interest(s), seeding the first cell population on the bottom side of the...

Materials

Name	Company	Catalog Number	Comments
For Cell Culture
AG01522 (i.e., AG1522) human diploid fibroblast	Coriell	107661	Passage 8-13
MDA-MB-231-luc-D3H1 breast adenocarcinoma cell line	PerkinElmer	119261	Parental line: ATCC (#HTB-26)
MDA-MB-231/GFP breast adenocarcinoma cell line	Cell Biolabs	AKR-201
Eagle's minimal essential medium (MEM)	Corning Cellgro	15-010-CV
Fetal Bovine Serum (FBS), Qualified	Sigma	F6178-500mL
Corning Glutagro Supplement (200 mM L-alanyl-L-glutamine)	Corning Cellgro	25-015-Cl
Penicillin Streptomycin Solution, 100x	Corning Cellgro	30-002-Cl
Transwell Insert (i.e., permeable microporous membrane insert) (0.4 μm pore)	Costar	3450
Transwell Insert (i.e., permeable microporous membrane insert) (1 μm pore)	Greiner bio-one	657610
Transwell Insert (i.e., permeable microporous membrane insert) (3 μm pore)	Costar	3452
6-well Culture Plate	Greiner Bio-One Cellstar	657160-01
75 cm2 cell culture flask	CellStar	658 170
Phosphate-Buffered Saline (PBS), 1x	Corning Cellgro	21-040-CV	without calcium & magnesium
0.25% (vol/vol) Trypsin, 2.21 mM EDTA, 1x	Corning Cellgro	25-053-Cl
15 ml Centrifuge Tube	CellTreat	229411
35 mm x 10 mm Cell Culture Dish	Greiner bio-one	627 160
Name	Company	Catalog Number	Comments
For Immunofluorescent Microscopy
Mouse anti-Caveolin 1	BD Transduction Laboratories	610406	In situ Immunofluorescence - 1:5,000
Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate	ThermoFisher Scientific	A-11029	In situ Immunofluorescence - 1:2,000
Bovine Serum Albumin - Fraction V	Rockland	BSA-50	Immunoglobulin and protease free
16% (wt/vol) Formaldehyde Solution	ThermoFisher Scientific	28908	Dilute to 4% with 1x PBS
Premium Cover Glass (22 mm x 22 mm No.1)	Fisher	12548B
Triton X-100	Sigma	T8787-50ML
SlowFade Gold antifade reagent with DAPI	Invitrogen	S36938
Name	Company	Catalog Number	Comments
For Flow Cytometric Analysis
Calcein, AM	Molecular Probes	C3100MP
Hanks' Balanced Salt Solution (HBSS)	Gibco	14025-076
Name	Company	Catalog Number	Comments
For Western Blot Analysis
Mouse anti-Caveolin 1	BD Transduction Laboratories	610406	Western Blot - 1:1,000
Tween-20	BioRad	170-6531
Nitrocellulose Membrane (0.2 μm)	BioRad	162-0112
Western Lightning Plus-ECL	PerkinElmer	NEL104001EA
BioRad DC Protein Assay	BioRad	500-0116
Sodium dodecyl sulfate (SDS)	BioRad	161-0302
Sodium deoxycholate monohydrate (DOC)	Sigma	D5670
IGEPAL CA-630 (NP40)	Sigma	I8896
30% Acrylamide/Bis Solution, 37.5:1	BioRad	161-0158