Utilization of discarded surgical tissue from ultrasonic aspirators to establish patient-derived metastatic brain tumor cells: A Guide from the Operating Room to the Research Laboratory

Abstract

Patient-derived cells from surgical resections are of paramount importance to brain tumor research. It is well known that there are cellular and microenvironmental heterogeneity within a single tumor mass. Thus, current established protocols for propagating tumor cells in vitro are limiting because resections obtained from conventional singular samples limits the diversity in cell populations and does not accurately model the heterogeneous tumor. Utilization of discarded tissue obtained from CUSA of the whole tumor mass allows for establishing novel cell lines in vitro from the entirety of the tumor; thereby creating an accurate representation of the heterogeneous population of cells originally present in the tumor. Furthermore, while others have described protocols for establishing patient tumor lines once tissue has arrived in the research lab, a primer from the OR to research lab has not been described before. This is integral as basic research scientist need to understand the surgical environment of the OR including methods utilized to obtain a patient’s tumor resection in order to more accurately model cancer biology in laboratory.

Basic Protocol 1:

Establishment of Brain Tumor Cell Lines from Patient Derived CUSA Samples

Basic Protocol 2:

Selection of tumor cells in vitro

Introduction

Brain tumors are a diverse group of neoplasms arising from different cells within the central nervous system (CNS) or from systemic cancers that have metastasized to the CNS. Overall, brain metastases are the most common intracranial tumors in adults, accounting for significantly more than one-half of brain tumors. Systemic tumors which metastasize to the CNS include melanoma, breast and lung cancer [1, 2]. Adult primary brain tumors include astrocytic, oligodendroglial, ependymal, and meningeal tumors. Pediatric brain tumors can include gliomas (low-grade, malignant, brainstem, and optical) and medulloblastoma.

The brain tumor microenvironment is an integral mediator of cancer progression in primary and metastatic brain malignancies. In addition, the central nervous system is one the most complex biological systems, which poses unique obstacles but also harbors opportunities for discovery [1, 3–8]. Therefore, patient-derived in vitro tumor cultures serve as an integral initial point for elucidation of brain tumor biology. They are an excellent tool to study genetics, drug targets, and efficacy of radiation and chemotherapies in cytotoxicity of cancer cells.

Currently, there are many available brain tumor cell lines (primary and metastatic) which are in research use. However, most of these cell lines have been in use for a number of years and suffer from genetic and phenotypic drift. Importantly, these cancer lines were derived from a single site within the original tumor mass; thereby reducing their heterogeneity – an important feature of tumor cells. While others have described protocols for establishing patient tumor lines, a primer from the operating room (OR) to lab has not been described before. Thus, institutions with access to samples from the operating room should consider establishing their own cell lines in order to facilitate clinically relevant research. This protocol describes sample preparation from generally discarded tissue obtained from ultrasonic aspirators which is more heterogenous as the tissue is collected from various parts of the tumor rather than from a resected piece which was segmented and given to the research lab. Establishing brain tumor lines from heterogeneous tissues provides a more accurate recapitulation of the tumor.

Patient-derived tumor cell lines are an essential research tool for cancer biologists. Although there are many established cell lines from a variety tumor subtypes, a majority fail to accurately characterize the tumor in humans. Cultured cells tend to derived from a small resection biopsy which can result in large genetic and phenotypic drift, reducing the heterogeneity of tumors – a feature of tumors readily seen in the clinic. As such, it is important to establish cell lines derived from patient samples to accurately recapitulate the diversity of cancer genetics within the laboratory. Utilizing whole tumor samples from cavitron ultrasonic surgical aspirator (CUSA) allows researchers to capture a heterogeneous tissue sample in hopes of establishing a cell line that represents the overall tumor. Thus, the ability to accurately model tumor phenotype in vitro from the patient’s tumor resection from the operating room (OR) will lead to increased ability to perform translational research which can then readily be correlated to clinical outcomes.

In this protocol, we describe how to acquire the tumor sample from the OR and process them in the laboratory, and how to establish pure cancer cell cultures in vitro. In the supporting protocols, we describe how to sterilize equipment, and how to coat tissue culture dishes with specific extracellular matrices to facilitate cell attachment.

Basic Protocol 1

Establishment of Brain Tumor Cell Lines from Patient Derived CUSA Samples

Introduction

The purpose of the following methods is to establish brain tumor cells lines acquired through CUSA samples from brain tumor resections. Upon receiving the sample, it is processed in sterile conditions and put into cell culture flasks. Flasks are monitored for two weeks to determine which cell types (i.e. fibroblast, tumor cells) are present and whether they are proliferating. In this protocol, we will describe the initial phase of this process – establishing viable cells in vitro from CUSA samples.

Materials

Laminar Flow Cabinet/ PCR Cabinet

Standard Light Microscope

Sterile Surgical Tools (Spring Scissors, Standard Forceps)

Sterile 1x Phosphate Buffered Saline

4% Formaldehyde

Sterile 10cm tissue culture plates

Sterile 15mL conical tubes

Sterile 50mL conical tubes

Sterile 10mL pipettes

Portable Pipette Controller

Pasteur Pipette

Cryovials

Centrifuge

0.05% Trypsin

Cell Incubator

Rotating plate shaker

Patient Derived Lesion (PDL) Medium

Collagen Coated T25 or T75 plates

Protocol 1: Processing the Brain Tumor Sample - From the OR to the Lab

1. The Institution, operating physician, and laboratory scientist should have a current IRB that allows for patient tissue to be used for research purposes. It is important to maintain quality rapport with operating physician to facilitate logistics between hospital staff and laboratory staff in acquiring the specimen.

2. Laboratory Scientist, who have gone through IRB and HIPPAA training, can inquire from hospital neurosurgery coordinator to access operating room (OR) schedules in order to find patient with desirable tumor sample.

3. Laboratory scientists discuss with neurosurgeon and inquire if tumor sample can be obtained.

4. Upon receiving approval, laboratory scientist waits for tumor sample availability the day of surgery after consent has been received from patient.

5. OR technician receives tumor sample from surgeon and places directly in sterile saline solution. OR contacts laboratory scientist as soon as possible and informs them that the tumor is available.

6. Scientist goes to OR, makes note of location where lesion was excised, intraoperative tumor characteristics, vascularity of lesion, extent of invasion (encapsulated, presence of clean tissue planes, etc.), and patient’s medical record number (MRN).

7. Tumor sample obtained from ultrasonic aspirators (Figure 1, 2) is placed on ice and sample is transported back to the laboratory.

8. Dissection station is prepared as shown in Figure 3.

9. Make PDL medium in which cells will be cultured according to the recipe below. Medium can be made previously and stored in 4°C. PDL medium contains 20% Fetal Bovine Serum (FBS) and 2% Glutamine to ensure maximal amount of nutrients for cells.

   1. 100mL Advanced DMEM/F12 (Gibco Catalogue #: 12634-010)
   2. 100mL Neurobasal®-A Medium (Gibco Catalogue #: 10888-022)
   3. 50mL FBS
   4. 5mL GlutaMAX™-I (Gibco Catalogue #: 35050-061)
   5. 2.5mL Anti-Anti (Gibco Catalogue #: 15240-062)

10. 45 mL (or enough PBS to fully submerge tumor sample to avoid dehydration of sample) of sterile 1x Phosphate Buffered Saline (PBS) is poured into 10cM tissue culture plate and tumor sample is placed into PBS. Ensure tissue is immersed in PBS throughout the entire process. (Figure 4)

11. Label cryovials and 15 mL conical tube (filled with 4% formaldehyde) with appropriate name to identify sample. Ensure that patient MRN is not associated with the given sample name to ensure patient confidentiality.

12. Using dissection microscope, sample is separated into 4 smaller samples to be used for:

    1. qPCR analysis
    2. Protein analysis
    3. Tissue sections for staining applications
    4. Cell culture

13. Size of 4 smaller samples is up to the discretion of the scientist to best fit their studies, however, it should be noted that ideal tissue (i.e. non-cauterized, devoid of large concentration of blood vessels) should be used for establishing cells in vitro.

14. Using forceps (or a tissue scooper) place sample in cryovials and 15 mL conical tube for fixation, leaving only sample to be used for cell culture in the 10cm dish.

15. Of the remaining sample, confirm that tissue is minced and is approximately 1mm³ in size.

16. Using a sterile 10mL pipette, moisten the pipette with sterile PBS. If this process is not done, tissue chunks will adhere to the side of the pipette.

17. Place the moistened 10mL pipette into the 10cm dish and remove sample containing PBS and place into new 50mL tube.

18. Spin tube in centrifuge for 5 minutes at 1000 RPM. Carefully remove supernatant.

19. Resuspend cell pellet in 5mL 0.05% Trypsin and incubate in sterile cell incubator (37°C, 5% CO₂) for 10 minutes on a rotating plate shaker.

20. Remove 50mL tube from incubator. To maximize single cell suspension, agitate suspension by titurating the sample with a Pasteur pipette and a portable pipette controller. Duration of tituration should be optimized based on the sample.

21. Add 5mL sterile FBS containing medium to counteract effects of trypsin and to reduce cell clumping/death.

22. Allow any remaining chunks to settle (approximately 30 seconds to 1 minute), and remove supernatant (approximately 9 mL), careful to not disturb the settled tissue chunks, and place in new 15mL tube. This process enhances presence of single cell suspended cells.

23. Add 10mL PDL Medium to tissue chunks and plate in collagen coated T25 tissue culture flask. Collagen coating will be explained in Support Protocol 2. Place flask in cell incubator and monitor for media color change.

24. Centrifuge single cell suspension for 3 minutes at 1000 RPM. Remove supernatant and replace with 10mL of PDL medium.

25. Plate cells in T25 tissue culture flask and place in cell incubator. Monitor cells for media change but avoid disturbing flask for at least 3 days to enhance cell attachment.

Figure 1-.

An example of an ultrasonic tissue ablation system (CUSA Clarity) which is utilized in the operating room (Left). The attached precision handpiece is utilized in neurosurgery to safely and effectively resect tumors ranging from soft to firm consistencies, which includes removal of primary and metastatic malignancies (Right).

Figure 2 –

Example of a tissue trap contained within the collection system of a CUSA. The aspirate enters through tubing from handpiece previously described in Figure 1, with the porous bag trapping morselized tumor and allowing liquid to pass through. The bag is cut open (magnified segment) to visualize tumor tissue which will be used for establishment of brain tumor cell lines. Various different tissue traps are available for the ultrasonic aspiration systems.

Figure 3 –

Tissue Dissection Hood setup: 1) Cryopreservation tubes for genomics studies, 15mL conical tube with formaldehyde for tissue preservation, and 50mL tubes with sterile 1x PBS to submerge tissues. 2) Autoclaved tools, including forceps and scissors. 3) Microscope for close observation of tissue dissection. 4) 10mL pipettes and a portable pipette controller. 5) Sterile 10cm dish.

Figure 4 –

Example of discarded tissue from CUSA once removed from the OR mesh bag placed in PBS.

Support Protocol 1

Sterilization of Microsurgical Tools in Preparation for Dissection

Introduction

The aim of this protocol is to aid in the prevention of contaminated of cell culture flasks due to unsterilized microsurgical tools. It is already a difficult task to avoid contamination transferring a tissue sample from the OR to the laboratory. Although the OR is sterile, the level of cleanliness does not mirror the cleanliness of a laboratory hood. We can merely try our best to avoid contamination during the time. However, contamination of cell cultures can easily occur through dirty dissection tools. Thus, through this protocol, we aim to show how sterilization of contaminated tools, a variable controllable by the scientist, is an important process in establishing cell cultures from brain tumors.

Materials

Wesodyne

Metal Container

Autoclave Bags

Autoclave

Protocol Steps

1. Dilute Wesodyne according to manufacturer specifications.

2. Fully submerge soiled microdissection tools in diluted Wesodyne for 15 minutes.

3. Rinse tools with ddH₂0 and allow to dry.

4. Place dissection tools in autoclave bag. Make sure to place handles towards the side that will be opened to avoid contaminating the side of the tool which will be in contact with the tissue.

5. Autoclave tools.

Support Protocol 2

Collagen Coating of Tissue Culture Flasks

Introduction

Establishing tumor cell lines from patient derived tumor samples is a difficult task because it requires the cell to acclimate to a two-dimensional growth condition which does not have all the nutrients that were originally present in the three-dimensional tumor microenvironment. Thus, to model a three dimensional environment, it is crucial to provide an extracellular matrix for cells naïve to culture conditions. This process helps increase cell attachment which is vital for establishing cell cultures from tumor samples. Here we detail a protocol for Collagen coating for tumor cell derivation. While we describe Collagen as a source for coating, other extracellular matrix basement membranes (i.e. Polyornithine Fibronectin or Poly-L-Lysine) coating could be utilized as previously described [9, 10].

Materials

Collagen I, Rat Tail (Gibco A10483–01, 20mg at 5mg/mL)

T25/T75 Tissue Culture Flasks

Glacial Acetic Acid (17.5M)

Sterile ddH₂0

Filter

Protocol Steps

1. Prepare 0.02M acetic acid by pipetting 571 μL of stock acetic acid into 500mL of ddH₂0 and filter the solution.

2. Dilute collagen to 100μg/mL in 0.02M acetic acid at the final volume needed.

   1. T75 – 4mL/plate
   2. T25 – 2mL/plate

3. Add solution to plates and let incubate inside cell culture hood at room temperature for 2 hours.

4. Carefully aspirate solution from flask.

5. Rinse dish 1 time with equal volume PBS to ensure removal of acetic acid. Remove as much solution as possible by holding flasks at a slight angle for one minute.

6. Plates should then be sealed, and can be stored at 4°C.

Basic Protocol 2

Selection of tumor cells in vitro

Introduction

While Basic Protocol 1 focuses on deriving viable cells that can be grown from CUSA samples, establishing a pure tumor population requires further techniques. CUSA samples can contain a multitude of different cells, including support/stromal cells like fibroblasts and microglia, in conjunction to tumor cells. Stromal cells can overtake cultures, essentially suffocating the tumor cells, which take longer to become acclimated to in vitro conditions. Thus, it is imperative to use the techniques described below to establish pure tumor cell populations without sacrificing viability of these cells. Cells from the CUSA sample which have adhered to the plate will be lifted, placed in new flasks, and cultured in sphere forming conditions on a rotating plate shaker inside the incubator. This method helps eradicate supporting cells which do not survive in such conditions (i.e. support cells) – thus, forming a pure tumor cell population. Moreover, fluorescence activated cell sorting (FACS) can be utilized for sorting the heterogeneous cell population. Referencing pathology reports on tumor subgroup status may present cell surface markers which can be used for sorting tumor cells to create a pure tumor cell population.

Materials

0.05% Trypsin

1x Sterile PBS

15 mL conical tube

Centrifuge

Freezing Medium

Rotating Plate Shaker

Cell Incubator

Protocol Steps

1. Cells (all types) have sat down and appear to be proliferating (media color is changing).

2. Trypsinize cells (5mL) until all cells have been lifted from the culture surface.

3. Wash plate with equal volume 1x sterile PBS and place diluted trypsin in 15 mL conical tube.

4. Centrifuge for 5 minutes at 1000 RPM. Remove supernatant and resuspend pellet in 1mL PDL media. Ensure that no cell clumps are present.

5. Freeze down half of cells using desired freezing medium/technique.

6. Place remainder of cells in a non-collagen coated flask containing 15mL PDL medium.

7. Place plate on a rotating plate shaker that is inside the cell incubator. Stromal cells, such as fibroblast, tend to not survive in sphere forming conditions, while tumor cells maintain the ability to do so. Moreover, it is imperative to dilute the original cell population, as we have observed that if there are a lot of cells in floating conditions, stromal cells can survive by attaching themselves to tumor cell clumps.

8. After 5 days, move entire cell containing medium to a collagen coated T75 flask, and do not disturb flask for 2 days to promote cell attachment.

9. Some stromal cells may survive the initial round of tumor cell selection, as shown in Figure 5. Repeat steps 2–8 until a pure tumor population is achieved.

Figure 5 –

In vitro derived tumor cells from the CUSA discarded tissue. Black arrows point to the fibroblasts, which can quickly take over cultures. Red arrows show the cancer cells, which are morphologically different than astrocytes (Image take at 20x).

Support Protocol 1

FACS sorting tumor sample to isolate cancer cells from heterogeneous cell population

Introduction

If there is difficulty separating stromal cells from tumor cells, FACS can be used. Pathology reports can identify specific cell surface markers (i.e. Her2), which can be targeted using cell surface marker antibodies.

Materials

FACS Buffer

FACS Antibodies

FACS Machine

Protocol Steps

1. Trypsinize cells until all cells have been lifted from the tissue culture flask

2. Centrifuge cells at 1000 RPM for 5 minutes to pellet cells.

3. Resuspend cells in FACS buffer (10% FBS, 1% Sodium Azide, 1x PBS)

4. Wash cells twice with FACS buffer for 5 minutes

5. Pipette 80% of cell mixture into new eppendorf tube for staining

6. Remaining 20% of cell mixture will be utilized as a negative control for FACS analysis

7. Add conjugated antibody per manufacturer recommendation and incubate for 30 minutes in the dark.

8. Wash cells twice with FACS buffer and keep cells on ice until cell sorting.

9. Bring an Eppendorf tube with PDL medium to sort cancer cells directly into medium.

Commentary

Background Information

Ultrasonic aspirators have been a mainstay of the neurosurgical arsenal since 1978 [11]. They are used both alongside, and as alternatives to, other more traditional tumor debulking methods including steel instrumentation, suction, and loop cautery. Ultrasonic aspirators deliver high speed mechanical waves to soft tissues that are high in water content in order to disrupt them by breaking tissues into small fragments that are collected via suction [11]. The system function is functionally divided into three main functions: fragmentation, irrigation, and aspiration through a hand-held surgical device. First, fragmentation is defined as breaking up the tissues into small morsels. Irrigation is the process of transferring liquid to the hand piece, while aspiration is the suction of the fluid, fragmented tissue, and other debris in the surrounding area into a canister. The amplitude of each of these functions can be individually controlled based on surgeon preference, cortical location and, most importantly, pathology of the treated lesion. The device consists of a handpiece with a hollow tip that delivers the ultrasonic waves contained within an irrigator which cools the element, with an aspirator to remove the debris and irrigation. As ultrasonic aspirators work by inducing vapor pockets in water rich tissues, their benefit lies in the fact that they cause decreased damage to collagenous tissues, thus limiting (but not eliminating) risk to adjacent critical vascular structures that must be preserved. Handpieces vary in length, diameter, and straight or curved shape to adjust for tumor depth, size, and angle relative to surgical approach, respectively.

There are a variety of ultrasonic aspirators available on the market today through a number of different vendors; however they employ the same basic concepts, with some small variances that are mostly proprietary devices meant to differentiate their product, such as special tips, or the size and shape of the grip and handling apparatus. The Cavitron Ultrasonic Surgical Aspirator (CUSA), a system made by Integra (Figure 1), is the most commonly used, and will be the focus of this paper, though the SONOPET system made by Stryker is also widely used. There have been few studies directly comparing the two systems, although a review of the available literature concludes that they seem to be functionally interchangeable.

The ultrasonic aspirator handpiece is connected to a base system through which tumor fragments are suctioned into a collection chamber. Though the CUSA aspirate is typically discarded, the viable tumor sample present in the aspirate is potentially valuable for experimental purposes. In 1990, Oakes et al. proposed a method of collecting tumor fragments from the CUSA aspirate by stopping the suction line of CUSA with a sterile trap (eg. sputum collector) [12]. The trap was immediately then placed on ice and transferred to the laboratory for analysis. Oakes et al. were able to obtain clean, viable medulloblastoma cell lines through serial centrifugation (1000 rpm for 5 minutes) and red blood cell lysis procedure with ammonia chloride. The established cell lines were transplanted into athymic nude mice and the mice developed highly invasive and undifferentiated tumors after 1–6 months.

A separate study was also able to successfully collect tumor samples from a simple sterile suction trap that was connected to the CUSA suction hose; this method created a closed sterile environment to avoid contamination [13]. The tissue was then centrifuged, washed, and disassociated with trypsin for 30 minutes until no fragments were present. The cells were seeded as a monolayer in 10% FBS media. To determine the viability of the cells, a viability assay was performed in which after immunohistological experiments on the tumor cells after 15 passages were ensued. Their study was able to confirm that the cells isolated from the CUSA showed a cell viability of 67–82% and that the tumor cells also preserved their histological detail.

Therefore, the ability to harvest the heterogenous tumor phenotype in vitro utilizing the these ultrasonic aspirators from patient’s tumor resection in OR will lead to increased ability to perform translational research which can then readily be correlated to clinical outcomes.

Ultrasonic aspirators have become essential in surgical resection for a wide variety of tumors, both intracranial and intraspinal, including gliomas, meningiomas, and metastases [11, 14]. They can further be employed through both open craniotomies, approaching tumors through a standard cranial opening, as well as endoscopic approaches, through the nasal passage [14]. Ultrasonic aspirators are primarily used as a method of mechanical debulking in tumors that are not soft enough to be internally debulked by simple suction alone, but not so firm as to require sharp resection or cautery loop [15]. As noted above, amplitude of fragmentation is adjusted to increase both fragmentation rate and remove tissues with greater resistance to fragmentation. Tissues with weak intracellular bonds and high fluid content, including cortical parenchyma, are more easily fragmented, while tissues with stronger intracellular bonds such as vessel walls and tumor capsules are more difficult to fragment [16]. This difficulty of fragmentation dictates the tumor types in which it is most commonly employed, as gliomas often can be removed with suction or low amplitude fragmentation settings. Meningiomas with decreased firmness and most metastases have a consistency more amenable to ultrasonic aspiration. Local structures may also dictate how the instrument is employed, as most metastases and meningiomas have the “softer” core of the tumor removed via ultrasonic aspirator, with the firmer tumor capsule folded inwards and removed piecemeal or en bloc.

It can prove difficult, however, to determine what the most effective implement will be for a given tumor, and often, all of these surgical techniques are used in conjunction. A study by Zada et al. proposed a grading system for intracranial meningiomas based on consistency in order to standardize the characterization of meningiomas. Tumors can be characterized via neuroimaging, allowing surgeons to objectively select the most appropriate surgical tools preoperatively [15]. Ahn et al. showed that larger volume of a tumor is another significant factor that determines use of the CUSA [17].

In 1985, Oosterhuis et al. showed that 2% of all cells collected from CUSA aspirate were morphologically intact, and 1% were viable. Viability of tumor cells was not significantly affected either by the type of irrigation fluid used, even if the solution was ‘hostile’ such as deionized water or Dakin’s solution, or the output setting of the machine, and these cells could be successfully cultured both in vitro and in vivo [18]. While this finding proves that CUSA aspirate can be a legitimate source of tissue for basic science studies of brain neoplasms, it also implies a risk of seeding tumor with use of CUSA during surgery. In fact, a drawback of ultrasonic aspiration in brain metastases is the potential to increase the risk of leptomeningeal seeding, particularly when the tumor contacts the CSF pathway [17]. Such findings may deter surgeons from use of this device for metastatic tumors, particularly those in periventricular regions. Finally, another important consideration to include when receiving CUSA samples is the presence of adjacent morselized aspirate, including vasculature or cortical tissue. Bone fragments, in particular, are commonly intermixed in pituitary tumor samples that were procured through a transphenoidal approach [14].

Critical Parameters

For Basic Protocol 1, samples should be transferred to the laboratory as soon as possible. If processing of the samples cannot be done immediately, sample may be stored in PDL medium at 4°C overnight and processed the following day. When mincing the tissue, ensure that sample is fully submerged in saline/medium upon initial resection from patient to reduce drying of tissue. For Basic Protocol 2, it is important to isolate the tumor cells from the stromal cells. Stromal cells become acclimated to in vitro conditions quicker than tumor cells, and thus eventually overtake the culture population and reduce the viability to tumor cells.

Troubleshooting

For Basic Protocol 1, if you do not see viable cells in your flask after 3 days of incubation, it may be that the sample did not have viable cells, or the cells were not able to survive and adhere to the tissue culture flask. This process yields cell lines from approximately 20% of samples.

Understanding Results

Upon visualizing cells adhered to the tissue culture flask (Figure 5), it is important to view the morphology of the cells to determine whether the population is heterogeneous. As shown by the red and black arrows, cells of different backgrounds are present in the culture dish. Thus, further selection, as described in Basic Protocol 2, is necessary to select for tumor cells.

Time Considerations

Processing of the sample as described in Basic Protocol I takes approximately 30–45 minutes. Keep in mind that receipt of the sample may be delayed depending on the situation in the operating room. Sterilization of the tools should be done ideally the day before expected resection surgery. However, sterilizing tools a few hours beforehand will still work. Preparation of collagen-coated plates takes approximately 2.5 hours. Plates should already be made in anticipation of future tumor resection surgeries. When using FACS to sort for tumor cells, the overall process of staining and sorting cells will take approximately 2.5 hours.