Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

Summary

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

Abstract

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

Introduction

In 2001, a putative population of multipotent stem cells from adipose tissue was described in the journal Tissue Engineering ¹. These cells were given the name Processed Lipoaspirate or PLA cells due to their derivation from processed lipoaspirate tissue obtained through cosmetic surgery. The isolation method described in this article was based on existing enzymatic strategies for the isolation of the stromal vascular fraction (SVF) from adipose tissue ². The SVF has been defined as a minimally processed population of red blood cells, fibroblasts, endothelial cells, smooth muscle cells, pericytes and pre-adipocytes that have yet to adhere to a tissue culture substrate ^{2, 3}. Culturing of this SVF over time is proposed to eliminate many of these contaminating cell populations and result in an adherent, fibroblastic population. These fibroblasts have been identified in the literature for the last 40 years as being pre-adipocytes. However, our research group demonstrated that these cells possessed mesodermal multipotency and renamed the adherent SVF population as PLA cells. Subsequent studies by numerous other research groups have added to this potential, suggesting both endodermal and ectodermal potentials (for review see ⁴). Since that time, numerous additional terms for these cells have appeared in the literature. In order to provide some type of consensus, the term Adipose-derived Stem Cells or ASCs was adopted at the 2^nd annual IFATS conference. As such, the term ASC will be used in this article.

The protocol described in this article is a relatively simple procedure that requires standard laboratory equipment and uses simple reagents such as phospho-buffered saline, standard tissue culture media reagents and collagenase. It can produce large numbers of ASCs depending on the amount of starting adipose tissue volume and subsequent culture time. However, the processing of such a large amount of adipose tissue can present some physical issues that can be mitigated to a degree using this protocol. Furthermore, this protocol does require sterile tissue culture facilities and approved biosafety hoods, thereby necessitating the use of an approved tissue culture facility. This requirement can also decrease the utility of the ASC population in clinical applications unless they are isolated in good manufacturing practice (GMP)-approved facilities designed for the isolation and expansion of materials for clinical use. As an alternative, automated systems that can isolate ASCs in a closed system in the operating theatre would avoid this key issue and allow for the immediate use of ASCs without any need for subsequent in vitro expansion. To date, there are six automated systems that are available commercially for the isolation of cells from human tissue. These systems may make it possible to isolate a significant number of ASCs from large amounts of adipose tissue immediately following its harvest. These ASCs could then be reintroduced into the patient for a variety of regenerative purposes without the patient ever having to leave the operating room. In addition to this protocol describing the manual isolation of ASCs, a protocol for automated isolation of ASCs using the Celution System is also given in a companion article.

Protocol

The protocol shown here describes the manual isolation of ASCs from lipoaspirates obtained through cosmetic procedures using enzymatic digestion and differential centrifugation. This protocol was first published in the journal Tissue Engineering in 2001 ¹, where the resulting cells were called Processed Lipoaspirate Cells or PLA cells because of their isolation from lipoaspirates. However, the term PLA cell has now been replaced with the term Adipose-derived Stem Cells or ASCs to give the field some sort of conformity in terms of nomenclature. The cells isolated through this protocol have been shown by many to possess mesodermal potential both in vitro and in vivo (for review see ⁴). In addition, the ectodermal and endodermal potentials of the ASC have also been explored ⁴. The isolation technique is simple and straightforward and requires approximately one hour to complete. The resulting cells are cultured under standard tissue culture conditions. With culture time, the ASC population becomes more homogenous, comprised of fibroblastic cells that expand easily and show good viability over the long-term in culture.

Note: The following pieces of equipment that are required are listed at the end of this article. All glassware, media and reagents should be sterilized through autoclaving or filtered through 0.22 μm filters. Aseptic tissue culture techniques should be followed at all times. To ensure this, all materials placed in the biosafety cabinet should be sterilized by spraying with a 70% ethanol solution and wiping with clean paper towels.

1. Prior to Harvest, Prepare the Following:

1. Sterile 1X phospho-buffered saline (PBS) for the washing of the lipoaspirates. Prepare approximately 1 L of 1x PBS for every 100 ml of lipoaspirate. Autoclave the PBS and allow to cool to room temperature before use. As an option, antibiotic/antimycotic may be added to a final concentration of: 100 I.U./ml penicillin, 100 μg/ml streptomycin and 2.50 μg/ml amphotericin B once the PBS has cooled.
2. Sterile 0.075-0.1% collagenase type IA (from Clostridium histolyticum) for enzymatic digestion of the lipoaspirate. Concentration will depend on the quality and source of the collagenase - i.e. crude vs. purified. Other collagenase types may be used (e.g. collagenase type II or IV) at similar concentrations. Prepare in 1x PBS and sterile filter using a 1,000 ml filtration system with a 0.22 μm filter (Figure 1A). However, sterile glass bottles fitted with a bottle top filter may also be used. Prepare and use at room temperature. Collagenase solutions may be stored for the short-term at 4 °C until needed. Do not freeze the collagenase solution as it may lessen its activity.
3. Sterile Control Medium (CM) for collagenase inactivation and culturing of the ASC population. For 500 ml of CM, combine the following: 500 ml DMEM (4.5 g/L glucose, with L-glutamine), 50 ml fetal bovine serum (heat inactivated), 5 ml penicillin/streptomycin (10,000 IU penicillin, 10,000 μg/ml streptomycin). As an option, 5 ml amphotericin B (250 μg/ml amphotericin B) may also be added. Sterile filtration of this media is required if non-sterile reagents are used. Place the CM in a 37 °C water bath 30 min prior to use to warm the medium.
4. Sterile glassware and plasticware for isolation. Autoclave a 1,000 ml glass beaker and allow to cool to room temperature before use. In addition, have the following plasticware ready: aseptic serological pipettes (5 ml, 10 ml and 25 ml), 50 ml centrifuge tubes, and tissue culture-treated culture dishes.

2. Isolation of ASCs from Lipoaspirates

Most lipoaspirates are collected in an aspiration container (see Figure 1B) and may be comprised of three distinct layers: 1) an upper layer of oil due to the lysis of mature adipocytes, 2) a middle layer of adipose tissue, and 3) a bottom, liquid infranatant containing saline and contaminating cell types such as red blood cells (RBCs). The top oil layer may not be present in significant amounts. If present, it should be aspirated as oil contamination of the resulting ASC culture could affect the viability of the culture. The bottom infranatant should be removed prior to processing to minimize contamination of the ASC cultures with RBCs. This will leave the middle, adipose tissue layer, which will then be washed and enzymatically digested to liberate its cellular, stromal vascular fraction (SVF).

1. Washing the lipoaspirate: Open the lipoaspirate container in the tissue culture hood and carefully decant the lipoaspirate into a sterile 1 L glass beaker. Allow the lipoaspirate layers to separate until the adipose tissue layer is well separated (Figure 1B).
   1. Aspirate off the oil layer (if present) using a sterile, glass pipette and the bottom saline infranatant using a 10 ml serological pipette. This will remove the greater majority of contaminating red blood cells and saline.
   2. Assess the volume of the resulting adipose tissue layer and add sterile 1x PBS at an equal volume. Stir the aspirate with the pipette used for aspiration in order to mix the lipoaspirate with the PBS. Allow the two layers to settle. As noted above, the 1x PBS may be supplemented with antibiotic/antimycotics.
   3. Aspirate the infranatant using a 10 ml serological pipette.
   4. Continue to wash the adipose tissue fraction as outlined in steps 2.1.2. and 2.1.3. until the adipose layer has a yellow/gold color. Aspirate the infranatant one last time, leaving the adipose tissue fraction in the glass beaker. To ensure adequate removal of the infranatant, allow the lipoaspirate layers to settle for 5 min after the last wash and before the final aspiration.
2. Enzymatic Digestion of the lipoaspirate: Prepare sterile collagenase 1A solution as outlined above in a filter unit. Prepare a volume equal to that of the adipose fraction and sterile filter.
   1. Following filtration of the collagenase solution, discard the upper filter unit, carefully pour the washed adipose fraction into the collagenase solution and tightly close the bottle.
   2. Place the collagenase/adipose mixture in a 37 °C water bath and digest at 37 °C for 30 min. Gently swirl the collagenase/adipose mixture every 5-10 min and place back in the water bath. The adipose tissue layer should take on a "smoother" appearance (Figure 1B) as the digestion proceeds. Additional digestion times (i.e. up to 2 hr) can be used if the adipose tissue layer still appears to have solid pieces of fat within it.
3. Isolation of ASCs: Place the digested collagenase/adipose mixture back into the biosafety cabinet. Be sure to sterilize the filter unit with 70% ethanol prior to placing back in the cabinet.
   1. Pipette 25 ml aliquots of the infranatant containing the SVF into sterile 50 ml centrifuge tubes.
   2. Add 25 ml of CM to each tube. Allow the CM to inactivate the collagenase by incubating at room temperature in the cabinet for 5 min.
   3. Centrifuge for 10 min at 1,200 x g to collect the SVF as a pellet.
   4. In the biosafety cabinet, aspirate the supernatant from each tube. Ensure the top oil layer and any floating adipocytes are aspirated with this supernatant (Figure 1B).
   5. Combine the SVF pellets into one centrifuge tube using 30 ml CM and divide equally over two new 50 ml centrifuge tubes. Centrifuge as in step 2.3.3. and aspirate the supernatants from the two SVF pellets (not shown in Figure).
      OPTIONAL: If RBC lysis is desired, resuspend each SVF pellet in 10 ml of an RBC lysis buffer (160 mM NH₄Cl) and incubate at room temperature for 10 min. Centrifuge as in step 2.3.3. and aspirate the supernatants to yield the SVF pellets (not shown in Figure).
   6. Combine the two SVF pellets into one new centrifuge tube using 5-10 ml CM (Figure 1B).
   7. To filter out larger tissue particles, pipette the resuspended SVF pellet onto a 100 μm mesh filter placed on top of a new 50 ml centrifuge tube and allow to filter by gravity flow.
   8. Pipette aliquots of the resuspended (and filtered) SVF pellet containing the ASCs onto tissue-culture treated culture dishes. Supplement each dish with an appropriate volume of CM.
   9. Culture the cells in CM for 3-4 days at 37 °C, 5% CO₂ without change of media.

Following this initial culture period, the culture media may be aspirated and any contaminating red blood cells gently removed by washing with sterile 1XPBS. Replace with CM and continue to culture the ASCs as needed. The type of tissue culture dish used and how the resuspended SVF pellet is divided among these dishes will depend on the number of cells desired following conventional tissue culture.

3. Characterization of the ASC Population: Flow Cytometry and Multilineage Differentiation

Once isolated, characterization of the ASC population should be performed. Conventional approaches for this include flow cytometry to characterize the cell surface CD antigen profile of the cells and in vitro differentiation to confirm their multilineage differentiation capacity. While multiple lineages can be assessed in ASCs, this protocol outlines the differentiation of ASCs into cells of the adipogenic, osteogenic and chondrogenic lineages as a means of confirming multilineage mesodermal potentials ⁵.

In vitro multi-lineage Differentiation of ASCs

1. Osteogenic Differentiation of ASCs: Prior to induction, prepare Osteogenic Medium (OM) as follows: To one 500 ml bottle of DMEM (4.5 g/ml glucose) add 25.0 ml of FBS (5.0% final concentration) and 5.0 ml of penicillin/streptomycin (10,000 I.U/ml and 10,000 mg/ml final concentrations, respectively). To this, add the following osteogenic induction agents to give the indicated final concentrations: Dexamethasone (0.1 μM final), L-Ascorbic acid 2-phosphate (50.0 μM final) and β-glycerophosphate (10.0 mM final).
   1. Prior to induction, harvest and plate the cultured ASCs into the desired tissue culture dish so that a confluency of approximately 50% is achieved. For every experimental dish plated, plate an additional dish for a non-induced control. Culture overnight in CM.
      Note: 50% confluency is recommended in order to allow for continued proliferation that may occur over the course of induction
   2. On the day of induction, remove the medium and add the following: OM to the desired osteogenic experimental wells and CM to the control wells. The volume of media used will depend on the size of the tissue culture dish. For 12 well dishes, 1.0 ml of media per well is sufficient. For 6 well dishes, 2.0 ml of media per well is sufficient. For 100 mm dishes, 10.0 ml of media per dish should be used.
   3. Culture the cells for a minimum of 14 days with changes of the appropriate medium every 3-4 days. Highly osteogenic ASC populations may show signs of extracellular mineral deposition as early as 7 days induction.
   4. Osteogenic differentiation (i.e. extracellular mineral deposition) may be confirmed using a von Kossa histologic stain for calcium phosphate. This stain will produce a black-brown precipitate identifying the presence of extracellular calcium phosphate (see Figure 2).
      To stain with von Kossa reagent:
      1. Remove the OM from the experimental cells and the CM from the control cells
      2. Fix the cells for 60 min in 4.0% paraformaldehyde prepared in 1x PBS.
      3. Wash cells two times with 1x PBS.
      4. Incubate the cells with 2.0% silver nitrate (prepared in water) in the dark for 30 min.
      5. Remove the silver nitrate, wash two times with distilled water and air-dry the cells.
      6. Expose the cells to UV light for 60 min to develop the calcium phosphate precipitate.
      7. Wash the cells several times with 1XPBS.
      8. Counterstain the cell with hematoxylin if desired.

2. Adipogenic Differentiation of ASCs: Prior to induction, prepare Adipogenic Medium (AM) as follows: To one 500 ml bottle of DMEM (4.5 g/ml glucose) add 50.0 ml of FBS (10.0% final concentration) and 5.0 ml of penicillin/streptomycin (10,000 I.U/ml and 10,000 μg/ml final concentrations, respectively). To this, add the following adipogenic induction agents to give the indicated final concentrations: dexamethasone (1.0 μM final), 3-isobutyl-1-methylxanthine (IBMX - 0.5 mM final), indomethacin (0.2 mM final) and insulin (10.0 μM final).
   1. Prior to induction, harvest and plate the cultured ASCs into the desired tissue culture dish so that a confluency of approximately 80% is achieved. For every experimental dish plated, plate an additional dish for a non-induced control. Culture overnight in CM. Note: Adipogenic differentiation of ASCs appears to be more efficient with increased confluency.
   2. On the day of induction, remove the medium and add the following: AM to the desired osteogenic experimental wells and CM to the control wells. The volume of media used will depend on the size of the tissue culture dish. For 12 well dishes, 1.0.ml of media per well is sufficient. For 6 well dishes, 2.0 ml of media per well is sufficient. For 100 mm dishes, 10.0 ml of media per dish should be used.
   3. Culture the cells for a minimum of 14 days with changes of the appropriate medium every 3-4 days. Highly adipogenic ASC populations may show signs of lipid vacuole formation as early as 7 days induction.
   4. ASCs undergoing adipogenic differentiation will develop multiple lipid vacuoles that may be easily visualized under the light microscope. In addition, adipogenic differentiation may be confirmed using an Oil Red O histologic stain that will accumulate within these vacuoles (see Figure 2).
      To stain with Oil Red O:
      1. Remove the media from the experimental and control cells and fix them for 60 min using a 10% formal calcium fixative. To prepare this fixative, combine the following: 1.0 g of CaCl₂, 25.0 ml 16% paraformaldehyde (4.0% final) and 75.0 ml distilled water.
      2. Wash the cells two times with 1x PBS.
      3. Wash the cells briefly with 70% ethanol.
      4. Incubate the cells at room temperature for 5 min with the Oil Red O solution. To prepare the Oil Red O solution, dissolve 2.0 g Oil Red O powder in 50.0 ml 70% ethanol and 50.0 ml acetone.
      5. Wash the cells with 70% ethanol and then with tap water.
      6. Visualize the cells soon after staining due to fading of the stain with time.

3. Chondrogenic Differentiation of ASCs: Chondrogenic differentiation is achieved through a high-density culture technique referred to as "micromass culture". Prior to culture, prepare Chondrogenic Medium (ChM) as follows: To one 500 ml bottle of DMEM (4.5 g/ml glucose) add 5.0 ml of FBS (1% final concentration) and 5.0 ml of penicillin/streptomycin (10,000 I.U/ml and 10,000 μg/ml final concentrations, respectively). To this, add the following chondrogenic induction agents to give the indicated final concentrations: L-ascorbic-2-phosphate (50.0 μg/ml final), insulin (6.25 μg/ml final), transferrin (6.25 μg/ml final) and TGFβ1 (10.0 ng/ml).
   1. Harvest the ASCs and centrifuge for 5 min at 1,200 x g to pellet the cells. Count the cells to determine the density of the cell suspension.
   2. To prepare the micromass pellets, prepare two cell suspensions: one experimental suspension prepared in ChM at a density of 1 x 10⁷ cells/ml and one control suspension prepared in CM at a density of 1 x 10⁷ cells/ml.
      1. Place 10.0 μl "droplets" of the cellular suspension in individual wells of a multi-well dish. Allow to adhere for 2-3 hr at 37 °C.
      2. Gently overlay the plated droplets with either ChM or CM being careful not to dissociate the cells.
      3. Culture the cells for up to 14 days in ChM or CM with changes in medium every 3-4 days. Cells cultured in ChM will condense over this time to form a high-density micromass pellet with little to no cells in a surrounding monolayer. Control cells cultured will not undergo this condensation and many will be found as a monolayer.
      4. To confirm chondrogenesis, fix the pellets for 15 min in 4.0% paraformaldehyde (prepared in 1x PBS) at room temperature. Stain for the presence of sulfated proteoglycans using an Alcian Blue histologic stain.
         To stain using Alcian Blue:
         1. Wash the paraformaldehyde-fixed pellets once in 1x PBS.
         2. Incubate the pellets in 0.1 N HCl (pH 1.0) for 5 min to lower their pH.
         3. Remove the HCl and add 1% Alcian blue reagent (prepared in 0.1 N HCl, pH 1.0). Incubate for 30 min at room temperature.
         4. Remove the Alcian blue reagent and wash the pellets with 0.1 N HCl (pH 1.0) to remove excess stain.
         5. Additional washes using tap water may be used to reduce background staining.
         6. As an alternative, the micromass pellets may be harvested, fixed overnight in 10% formalin, embedded in paraffin and the sections stained for Alcian Blue.

4. Flow Cytometric Characterization of ASCs

Characterization of the cell surface CD antigen profile may be accomplished through conventional flow cytometry.

1. Preparation of ASCs for flow cytometry: Prior to analysis, prepare a Flow Cytometry Buffer (FCB) comprised of the following: 1.0% BSA, 2.0% FBS and 0.025% saponin prepared in 1x PBS.
   1. Harvest the ASCs and centrifuge the cells for 5 min at 1,200 x g to produce a pellet.
   2. Resuspend the cells in sterile 1XPBS and count the cells using a hemocytometer. Determine the cell density of the suspension.
   3. Divide the suspension up into aliquots of 5 x 10⁵ cells total and centrifuge to pellet.
   4. Remove the PBS supernatant and fix the cells for 60 min at -20 °C with an excess of ice-cold 70% ethanol. If necessary, these cells may be stored in this 70% ethanol in a -20 °C freezer until needed.
   5. Following fixation, carefully aspirate the ethanol and gently wash the pellet two times with an excess of FCB. To wash:
      1. Add an excess of FCB and gently resuspend the pellet.
      2. Centrifuge for 5 min at 1,200 x g to collect the cells as a pellet.
      3. Carefully aspirate the FCB supernatant.
      4. Repeat.
   6. Remove the final FCB wash supernatant from each aliquot and resuspend in 100 μl of FCB. Add the desired fluorochrome-conjugated primary antibody using the manufacturer's suggested dilution. Incubate the cells on ice with the antibody for 30 min.
      Note: If fluorochrome-conjugated primary antibodies are not available, non-conjugated primary antibodies may be used.
   7. For each fluorochrome used (e.g. FITC, PE), incubate an aliquot of cells with 100 ml FCB containing isotype-matched IgGs as a negative control.
   8. Incubate one aliquot in 100 ml FCB containing no antibodies as an additional control.
   9. Wash each aliquot of cell two times with FCB as detailed in step 4.1.5. Following the last wash, resuspend the aliquots in 100 ml of FCB and proceed with flow analysis.
      Note: If non-conjugated primary antibodies were used: Following this wash step incubate the aliquots on ice for 20 min with FCB supplemented with the appropriate fluorochrome-conjugated secondary antibody using the manufacturer's suggested dilution. Wash two times as outlined in step 4.1.5.
2. Flow analysis of ASCs: For flow analysis, a minimum of 10,000 events should be counted. Unstained and isotype-matched controls should be used to set gates by forward scatter (FSC) and side scatter (SSC). For this, the no antibody controls (i.e. unstained) of step 4.1.8 should be used in order to eliminate debris and dead ASCs. The isotype-matched IgG controls of step 4.1.7. should be used to establish areas of positive fluorescence.

Results

The protocol outline above describes a manual, enzymatic method for the isolation of an SVF from a large volume lipoaspirate sample. Within this SVF are numerous cell populations, including the ASC. Numerous studies propose that culturing this SVF under standard tissue culture conditions will select for an adherent fibroblast population likely to be composed mainly of the ASC type. Consistent with this, we have shown, using flow cytometry and immunofluorescence, that cultured SVF pellets become essentially free of the ...

Discussion

Adipose tissue for the isolation of ASCs can come in many forms: from solid pieces of tissue obtained through resection or lipoplasty to smaller pieces obtained through either syringe extraction or suction-assisted lipoplasty (i.e. liposuction ). Whether more SVF cells (and thereby ASCs) can be obtained from resected or aspirated adipose samples is unclear as conflicting studies have been presented ^{16, 17}. It is possible that either form of adipose tissue is more than suitable for the isolation of SVF...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagent
DMEM (Dulbecco’s Modification of Eagle’s Medium)	Mediatech Cellgro	10-013-CV	with 4.5 g/ml glucose, L-glutamine, sodium pyruvate
Penicillin/Streptomycin	Mediatech Cellgro	30-002-CI	10,000 IU/ml penicillin/10,000 μg/ml streptomycin
Amphotericin B	Mediatech Cellgro	30-003-CF	250 μg/ml amphotericin B
10X PBS (Phospho-buffered Saline)	Mediatech Cellgro	25-053-CI	without calcium, without magnesium
Trypsin/EDTA	Mediatech Cellgro	20-031-CV	0.25 % trypsin/2.21mM EDTA
Collagenase type IA (from Clostridium histolyticum)	Sigma	C2674	crude preparation; <125 collagen digestion units/mg solid
FBS (Fetal Bovine Serum) heat inactivated	Gemini Bioproducts	100106	USDA source, heat inactivated
10 ml serological pipettes	Genesee Scientific	12-104
25 ml serological pipettes	Genesee Scientific	12-106
50 ml polypropylene centrifuge tubes	Genesee Scientific	21-106
100 mm tissue culture dishes	Genesee Scientific	25-202
150 mm tissue culture dishes	Genesee Scientific	25-203
500 ml Stericup Filter Units	Millipore	SCGPU05RE	PES membrane, 0.22 μm pore
Cell strainers	FisherBrand	22-363-549	100 μm nylon mesh
dexamethasone - water soluble	Sigma	D-2915
L-ascorbic-acid 2 phosphate	Sigma	A-8960
β-glycerophosphate disodium salt	Sigma	G-9422	also known as glycerophosphate
insulin	Sigma	I-6634	made from bovine pancreas
indomethacin	Sigma	I-7378
apo-transferrin	Sigma	T-4382
TGFβ1	R&D Systems	240-B-002	recombinant human
Oil Red O	Sigma	O-0625
Alcian Blue	Sigma	A-5268
Silver nitrate	Sigma	S-0319
Hydrochloric acid	Fisher Scientific	A144
Paraformaldehyde	Fisher Scientific	30525-89-4	supplied as a 16 % stock
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Equipment Needed
Class II A/B Biosafety hood	Thermo Scientific		ensure hood has vacuum lines for aspiration
Benchtop centrifuge	Hermle Labnet	Z383	Swing-out rotor for 50 ml tubes required, capable of 1200xg
Water bath	Fisher Scientific Isotemp	S52602Q	5-10L capacity, capable of 37C
Automated Pipette Aids	Drummond Pipette Aid XL	4-000-105
CO2 Incubator	Thermo Scientific	Forma 310	direct heat or water jacketed