Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Summary

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

Abstract

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1−10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

Introduction

Tumor antigen-specific cytotoxic T-lymphocytes (CTLs) play a crucial role in the response to cancers through a process known as cancer "immune surveillance". Their anti-tumor functions are enhanced by immune checkpoint blockade antibodies such as CTLA-4 inhibitors and PD-1/PD-L1 inhibitors. In non-small cell lung cancer (NSCLC), anti-PD-1/PD-L1 therapies result in response rates ranging from 0%-17% in patients with PD-L1-negative tumors and 36%-100% in those expressing PD-L1. The robust responses to PD-1/PD-L1 blockade observed in melanoma and NSCLC are shown by evidence of improved overall response rate (RR), durable clinical benefits, and progression-free survival (PFS). Currently, anti-PD1 treatments are the standard of care in second-line NSCLC treatment with nivolumab regardless of PD-L1 expression and with pembrolizumab in patients expressing PD-L1 ≥1%. In first-line treatment, standard of care is pembrolizumab alone in patients with NSCLC expressing PD-L1 ≥50% and can be potentially enhanced with chemotherapy (platin and doublet drug depending on histologic subtype)^1,2.

However, such an approach to patient management is debatable³, since PD-L1 expression in tumor cells by immunohistochemistry (IHC) is probably not the most ideal companion biomarker. Others such as tumor mutation burden⁴ (TMB), microsatellite instability (MSI), and/or microbiota are possibly interesting in this setting either alone or in combination. NSCLC are known to be heterogeneous tumors, either spatially (from a tumor site to another one) or temporally (from diagnosis to recurrence). Patients with NSCLC are usually fragile, and iterative invasive tissue biopsies may be an issue. Indeed, re-biopsy rate at first progression ranges from 46%-84% depending on series, and successful re-biopsy (meaning with histological and full molecular analysis) ranges from 33%-75%. This means that 25%-67% of patients cannot receive a comprehensive re-biopsy analysis during first progression^5,6,7,8.

The advent of "liquid biopsies" has thus generated considerable enthusiasm in this particular setting, as it enables crucial reassessment of molecular alterations during disease progression by examining circulating free DNA (cfDNA) derived from circulating tumor cells (CTCs). These live cells are released from the tumor into the bloodstream, where they circulate freely. Although not routinely used, the analysis of CTCs appears to be highly promising in the case of molecular and phenotypic characterization, prognosis, and predictive significance in lung cancer (via DNAseq, RNAseq, miRNA and protein analysis). Indeed, CTCs likely harbor phenotypic characteristics of the active disease rather than the initial markers (detected on tissue biopsies at diagnosis). Furthermore, CTCs bypass the problem of spatial heterogeneity of the tumor tissue, which may be a crucial issue in small biopsies. Consequently, PD-L1 expression on CTCs may potentially shed light on the discrepancies derived from its use as a predictive biomarker using tumor tissue.

Recently, PD-L1 expression has been tested in CTCs of NSCLC. Almost all of the patients tested⁹ were PD-L1 positive, complicating the interpretation of the result and its clinical use. Overall, PD-L1-positive CTCs were detected in 69.4% of samples from an average of 4.5 cells/mL¹⁰. After initiation of radiation therapy, the proportion of PD-L1-positive CTCs increased significantly, indicating upregulation of PD-L1 expression in response to radiation¹¹. Hence, PD-L1 CTCs analysis may be used to monitor dynamic changes of the tumor and immune response, which may reflect the response to chemotherapy, radiation, and likely immunotherapy (IT) treatments.

To date, CTCs isolation and PD-L1 characterization rely on various methods such as anti-EpCAM-coated magnetic bead-based CTC capturing, enrichment-free based assay, and size-based^12,13 CTC capture assays. However, CTCs were only detected in 45%-65% of patients with metastatic NSCLC, thus limiting their ability to provide any information for more than half of metastatic NSCLC patients. In addition, CTC count was low in most of these studies using size-based approach¹⁰. Furthermore, this method has led to discrepancies such as the detection of CD45(-)/DAPI(+) cells with "cytomorphological patterns of malignancy" in the bloodstream of healthy donors. These concerns highlight the need for a highly sensitive method of CTC collection associated with immune-phenotyping of atypical CD45(-) cells from healthy whole blood using additional cancer biomarkers (i.e., TTF1, Vimentin, EpCAM, and CD44) in NSCLC.

Consequently, we evaluated a spiral microfluidic device that uses inertial and Dean drag forces to separate cells based on size and plasticity through a microfluidic chip. The formation of Dean vortex flows present in the microfluidic chip results in larger CTCs located along the inner wall and smaller immune cells along the outer wall of the chip. The enrichment process is completed by siphoning the larger cells into the collection outlet as the enriched CTC fraction. This method is particularly sensitive and specific (detection of around 1 CTC/mL of whole blood)¹⁴ and can be associated with customized immunofluorescence (IF) analyses. These tools will enable setting up of a positive threshold for clinical interpretation. A workflow is thus described that enables biologists to isolate and immunophenotype CTCs with a high rate of recovery and specificity. The protocol describes optimal use of the spiral microfluidic device to collect CTCs, the optimized IF assays that can be customized according to cancer type, and use of free open-source software for measuring and analyzing cell images to perform a semi-automatic numeration of the cells according to fluorescent staining. In addition, microscope multiplexing can be carried out depending on the number of fluorescent filters/markers available.

Protocol

Samples were prospectively collected within the framework of the CIRCAN ("CIRculating CANcer") cohort based at the Lyon University Hospital following patient written consent. This study was integrated into the CIRCAN_ALL cohort. The study CIRCAN_ALL was recognized as non-interventional by the CPP South-East IV dated 04/11/2015 under the reference L15-188. An amended version was recognized as non-interventional on 20/09/2016 under reference L16-160. The CIRCAN_ALL study was declared to the IT and freedom correspondent of the Hospices Civils de Lyon on 01/12/2015, under the reference 15-131. Blood collection was performed when physicians observed the earliest indication of tumor progression.

NOTE: Use all the reagents and materials outlined in Table of Materials with the respective storage conditions for pre-analytical sample preparation and immunofluorescence assay. Substituting reagents and/or modifying storage conditions could result in suboptimal assay performance.

1. Decontamination of Spiral Microfluidic Device

NOTE: Decontamination of the spiral microfluidic device is a requirement to remove all immunofluorescence background generated from bacteria contamination, explore the cytomorphology of CTCs, and be able to differentiate them from normal immune cells. The protocol is optimized for blood samples collected in K₂EDTA tubes within 6 h after blood sampling and enriched using the spiral microfluidic device in clean conditions. Using this assay for other types of samples (other biological fluids) may require additional optimization. This decontamination protocol should be done once per week.

1. Preparation of reagents
   1. Preparation of the diluent buffer
      1. Sterilize 20 mL of diluent additive reagent using a 0.22 µm syringe filter and add directly to 1 L of 1x phosphate buffer saline (PBS) ultra-pure grade (Table of Materials).
   2. Sterilization of reagents and the input straw
      1. Sterilize the RBC lysis buffer and resuspension buffer (RSB; Table of Materials) using a 0.22 µm syringe filter and stock in a new 50 mL polypropylene conical tube for each solution.
      2. Sterilize the input straw by incubating at room temperature (RT) for 1 h in the all-purpose cleaning reagent (Table of Materials). Transfer the straw to the bleach-based cleaning agent (Table of Materials) and incubate at RT for 1 h.
      3. Rinse the input straw twice with sterile PBS for 1 h each and store the sterilized input straw in a surgically sterile bag (Table of Materials).
2. Decontaminating the spiral microfluidic device
   1. Disinfection using the all-purpose cleaning reagent
      1. Disconnect the diluent bottle cap from the diluent port of the spiral microfluidic device by unscrewing the brown screw. Under a microbiological safety cabinet, transfer up to 250 mL of all-purpose cleaning reagent (Table of Materials) into a new empty bottle.
      2. Screw the diluent bottle cap and straw to the bottle of 250 mL all-purpose cleaning reagent under a microbiological safety cabinet. Attach this bottle to the diluent port of the spiral microfluidic device by screwing back the brown screw.
      3. Transfer up to 100 mL of bleach (1% final concentration; Table of Materials) to the waste container supplied in the run kit (Figure 1A).
      4. Load a new sterile input straw onto the spiral microfluidic device (Table of Materials) in the input port. Load a new 50 mL centrifuge tube in the input port. Load a new 50 mL centrifuge tube in the output port.
      5. Proceed to prime the spiral microfluidic device by clicking on Prime on the spiral microfluidic device (3 min). Remove the input tube after the prime is completed.
      6. Transfer up to 15 mL of all-purpose cleaning reagent to a new 50 mL centrifuge tube with a serological pipette under a microbiological safety cabinet and attach the tube to the input port of the spiral microfluidic device.
      7. Before starting the run, check that the solution is free of excessive bubbles. If bubbles are present, remove them by slow aspiration with a pipette.
      8. Load a decontamination microfluidic chip in the spiral microfluidic device. Run a Program 3 on the spiral microfluidic device by clicking on Run and selecting the Program 3 (31 min).
         NOTE: The Program 3 of the spiral microfluidic device enables a rapid enrichment of CTCs in 31 min.
      9. Continue on with the spiral microfluidic device's cleaning step using the remaining volume of the all-purpose cleaning reagent in the input tube.
      10. Discard the input tube after cleaning step is completed, leaving behind the input straw.
   2. Decontamination using the bleach-based cleaning agent
      1. Disconnect the all-purpose cleaning reagent bottle cap from the diluent port of the spiral microfluidic device by unscrewing the brown screw. Under a microbiological safety cabinet, transfer up to 250 mL of bleach-based cleaning agent (Table of Materials) in a new empty bottle (Figure 1A).
      2. Screw the all-purpose cleaning reagent bottle cap and straw to the bottle containing the Bleach-based cleaning agent under a microbiological safety cabinet. Attach this bottle to the diluent port of the spiral microfluidic device by screwing back the brown screw.
      3. Transfer up to 15 mL of bleach-based cleaning agent to a new 50 mL centrifuge tube input tube using a serological pipette under a microbiological safety cabinet. Load the 50 mL centrifuge tube input position. Load an empty tube in output position.
      4. Before processing the run, check that the sample is free of excessive bubbles and if any are present, remove the bubbles by aspirating them slowly with a pipette.
      5. Run Program 3 by clicking on Run and selecting the Program 3 (31 min). After the run, proceed directly to the cleaning step using the remaining volume of bleach-based cleaning agent in the input tube.
      6. Discard the input and output tubes.
   3. Rinse the spiral microfluidic device.
      1. Disconnect the bleach-based cleaning agent bottle cap from the diluent port of the spiral microfluidic device by unscrewing the brown screw. Under a microbiological safety cabinet, transfer the straw from the bottle containing the bleach-based cleaning agent to the new bottle containing the diluent buffer. Screw the bottle to the spiral microfluidic device.
      2. Transfer up to 15 mL of sterilized water (Table of Materials) to a new 50 mL centrifuge tube input tube using a serological pipette under a microbiological safety cabinet. Load the 50 mL centrifuge tube input position. Load an empty tube in output position.
      3. Before processing the run, check that the sample is free of excessive bubbles and if any are present, remove the bubbles by aspirating them slowly with a pipette.
      4. Run Program 3 by clicking on Run and selecting the Program 3 (31 min). After the run, proceed directly to the cleaning step using the remaining volume of sterilized water in the input tube.
      5. Discard the input and output tubes.

2. Maintenance to Keep the Spiral Microfluidic Device Bacteria-free

NOTE: The routine maintenance should be done at the end of the day during the last cleaning step.

1. Transfer up to 7 mL of bleach-based cleaning agent into the new 50 mL centrifuge tube using a serological pipette under a microbiological safety cabinet. Screw the bleach-based cleaning tube in input port of the spiral microfluidic device.
2. Before processing the clean run, check that the input sample is free of excessive bubbles and if any are present, remove the bubbles by aspirating them slowly with a pipette.
3. Run the clean on the spiral microfluidic device.

3. Pre-analytical Enrichment of CTC from Patient Blood Samples

1. Collect 7.5 mL of blood in the K₂EDTA tube and keep under gentle agitation to avoid cell sedimentation and clotting. Process within 6 h.
   NOTE: If the blood is collected in a cell-free DNA blood collection tube containing preservative, store at 4 °C until processing. Ensure that the blood sample, RBC lysis buffer, and RBS buffer are at RT before proceeding with the enrichment step.
2. Transfer up to 7.5 mL of whole blood to a new 50 mL centrifuge tube input tube using a serological pipette under a microbiological safety cabinet.
3. Centrifuge at 1,600 x g for 10 min at RT. Collect the plasma fraction with a pipette without disturbing the buffy coat. Replace the plasma fraction by adding directly equivalent volume of PBS up to 7.5 mL.
4. Gently add RBC lysis buffer (Table of Materials) to blood sample to a final volume of 30 mL (for a K₂EDTA tube) or 37.5 mL (for a cell-free DNA blood collection tube). Gently invert the blood collection tube 10x and incubate for 10 min at RT.
   NOTE: The blood sample turns darker red during RBC lysis. If no change (from dark red and opaque) is observed after 10 min, gently invert the tube 3x and leave to stand for another 5 min maximum. Do not leave the sample in RBC lysis buffer for more than 15 min because it can compromise the sample quality and assay performance.
5. Centrifuge the lysed blood sample at 500 x g for 10 min at RT, with centrifuge brakes on (or highest deceleration speed). Use a Pasteur pipette or serological pipette to gently remove the supernatant until the volume reaches the 4-5 mL mark. Then, use filtered micropipette tips to remove the remaining supernatant.
6. Using a P1000 micropipette with a filtered tip, add 1.0 mL of RSB to the wall of the 50 mL centrifuge tube input tube. To avoid introducing bubbles into the mix, resuspend the cell pellet by gently pipetting up and down until the sample is homogeneous.
7. Add an additional 3 mL of RSB to the wall of the 50 mL centrifuge tube input tube (total volume 4 mL). Avoid introducing bubbles into the mix. Gently mix the cell suspension by gently pipetting up and down.
   NOTE: In the unlikely event that regular pipetting is unable to break down cell clumps (defined by being visible or blocking the pipette tip), filter the sample through a 40 μm cell strainer to remove any clumps. Add 150 μL of RSB to the sample to make up for volume loss from filtering. Note that this method is to be used sparingly and only when large clumps are observed.
8. Before proceeding to the enrichment step, check that the sample is free of excessive bubbles and if any are present, remove the bubbles and take care not to discard any sample. If tiny bubbles are present, their removal is not required.
9. Process the sample on the spiral microfluidic device.

4. Enrichment of CTCs from Patient Whole Blood with the Spiral Microfluidic Device

1. Load a new spiral microfluidic chip. Load two empty 50 mL centrifuge tubes in input and output ports.
2. Run a prime by clicking on Prime on the spiral microfluidic device (3 min). Remove the input and output tubes and load the sample to be processed in input port.
3. Load a clear 15 mL conical tube in output port to collect enriched CTCs. Run Program 3 by clicking on Run and selecting the Program 3 (31 min).
4. Unload the output tube and centrifuge at 500 x g for 10 min (acceleration: 9; deceleration: 5). With a 5 mL serological pipette, remove supernatant stopping at the 2 mL mark on the conical 15 mL tube. With a micropipette, remove supernatant stopping at 100 µL mark on the conical 15 mL tube. Process the enriched sample directly for immunofluorescence staining.

5. Immunofluorescence Staining

1. Enumerate on a chambered slide with a hemocytometer-type grid the number of cells per mL. Dilute the enriched sample with 0.2% anti-binding solution (Table of Materials) to a concentration reaching 100,000 cells/100 µL per cytospin.
2. Moisten the contour of the sample chamber using cotton (Table of Materials) with 50 µL of 0.2% anti-binding solution. Place a polylysine glass-slide in the sample chamber and close.
3. Coat a tip with 0.2% anti-binding solution by pipetting up and down 3x. Resuspend the enriched sample and transfer the cell solution into the sample chamber. Centrifuge with a dedicated centrifuge (Table of Materials) at 400 rpm for 4 min (acceleration low).
4. Place a silicon isolator around the area of deposition. Let dry the glass-slide under a microbiological safety cabinet for 2 min.
5. Prepare the fixation solution by diluting 1 mL of 16% paraformaldehyde (PFA) with 3 mL of sterile PBS. Add 100 µL of fixation solution (4% PFA) per sample and incubate at RT for 10 min. Remove fixation solution and perform three washes with 200 µL of PBS and incubate at RT each for 2 min.
   CAUTION: Use PFA under a chemical safety cabinet to prevent inhalation.
6. Prepare the saturation solution by diluting the fetal bovine serum (FBS) at 5%, Fc receptor (FcR) blocking reagent at 5%, and bovine serum albumin (BSA) at 1% in sterile PBS (Table of Materials). Add 100 µL of saturation solution per sample and incubate for 30 min at RT. Remove saturation solution.
7. Add 100 µL of antibody solution per sample (CD45 antibody 1/20; PanCK antibody 1/500; PD-L1 antibody 1/200; Qsp saturation solution 100 µL) (Table of Materials). Place the polylysine glass-slide in a 100 mm x 15 mm Petri dish. Moisten an absorbent paper with 2 mL of sterile water and close the Petri dish with the lid. Place at 4 °C overnight and protect from the light.
8. Remove the antibody mix and perform 3 washes with 200 µL of PBS incubating each wash for 2 min. Let the sample dry for 5 min and protect it from the light. Place 10 µL of mounting solution (Table of Materials) in the area of deposition and cover with a microscope coverslip without making a bubble. Seal the coverslip with nail polish.

6. Acquisition of Immunofluorescent Images with Straight Fluorescent Microscope and Associated Software

1. Use a straight fluorescent microscope with an X/Y motorized platform. Use a 20x objective to take 8-bit RGB tiff images in four channels corresponding to DNA dye (4',6-diamidino-2-phénylindole [DAPI]), PanCK dye (fluorescein isothiocyanate [FITC]), PD-L1 dye (CY3), and CD45 dye (CY5). Turn on the mercury lamp 15 min before use, and adapt the microscope and associated software to semi-automatized shoot.
2. Place the glass-slide on the platform.
3. In acquisition menu, define the four channels and set up the exposure time (DAPI: 15 ms, FITC [PanCK]: 500 ms; CY3 [PD-L1]: 800 ms; CY5 [CD45]: 1,000 ms). Define the tiles to scan. Click Tiles. In Advanced experiment, define the area to scan.
4. Adjust the focus on the screen. Click Start experiment.
5. Export TIF files of each channel and specifically name the image file with this information: Sample_NumberofTilesRegion_dye_NumberOfSubtiles.tif (e.g., Sample1_TR1_c1m01). Name dye as follows: the DAPI channel is c1, FITC channel is c2, CY3 channel is c3, and CY5 channel is c4.

7. Analysis of Immunofluorescent Images with Image Analysis Software

1. Download and install the free image analysis software from the Broad Institute website. Accept all default during installation. Open the image analysis software and click File | Pipeline from file | Analysis_4channels_CTC.cppipe.
   NOTE: The pipeline converts RGB color images into grayscale, removes artifacts by smoothing images with a median filter, identifies nuclei and cytoplasm, quantifies fluorescence intensities of each channel, and exports them into an excel file.
2. Drop files in the file list. Update the metadata to group the files by tiles.
   NOTE: All the instructions to group images are specified in the software. Name files appeared in NamesAndTypes module and files are grouped according to the number of the tile and channel per samples.
3. Click View Output settings and specify a correct default output. Click Analyze Images. Open the spreadsheet file corresponding to measure_intensity parameters.

Results

The first pre-requisite was to obtain uncontaminated (infectious agent-free) collections of CTCs for tissue culture and avoid IF background generated. The decontamination protocol enabled cleaning of all the pipes and pumps, and it resulted in the collection of CTCs with a good recovery rate without bacterial contamination. The enriched samples were compared without and with the decontamination protocol workflow of the spiral microfluidic device. To validate the decontamination protocol, the A549 cell line was used in ab...

Discussion

Two major points were raised in the present study, the first with regards to performance of the workflow for its transfer to clinical applications, and the second concerning the decrease in subjectivity for the analysis of fluorescence images obtained.

A performant and optimized workflow for CTC enumeration was initially determined using customizable IF assay after cell enrichment via a CTC label-free microfluidic system (spiral microfluidic device). Using this workflow, a pilot study confirme...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4',6-diamidino-2-phénylindole (DAPI)	Ozyme	BLE 422801	Storage conditions: +4 °C
BD Facs Clean – 5 L	BD Biosciences	340345	Bleach-based cleaning agent. Storage conditions: Room temperature
Bleach 1% Cleaning Solution 100 mL	Biolidics	CBB-F016012	Bleach. Storage conditions: Room temperature
Bovine Serum Albumin (BSA) 7.5%	Sigma	A8412	Storage conditions: +4 °C
CD45 monoclonal antibody (clone HI30) Alexa Fluor 647	BioLegend	BLE304020	Storage conditions: +4 °C
CellProfiler Software	Broad Institute		Image Analysis Software
Centrifuge device	Hettich	4706	Storage conditions: Room temperature
Centrifuge tube 50 mL	Corning	430-829	Storage conditions: Room temperature
Centrifuge Tube 15 mL	Biolidics	CBB-F001004-25	Storage conditions: Room temperature
ClearCell FX-1 System	Biolidics	CBB-F011002	Spiral microfluidic device. Storage conditions: Room temperature
Coulter Clenz Cleaning Agent – 5 L	Beckman Coulter	8448222	All-purpose cleaning reagent. Storage conditions: Room temperature
CTChip FR1S	Biolidics	CBB-FR001002	Microfluidic chip. Storage conditions: Room temperature
Cytospin 4	ThermoFisher	A78300003	Storage conditions: Room temperature
Diluent Additive Reagent – 20 mL	Biolidics	CBB-F016009	Storage conditions: +4 °C
EZ Cytofunnels	ThermoFisher	A78710003	Sample chamber with cotton. Storage conditions: Room temperature
FcR blocking Agent	Miltenyi Biotec	130-059-901	Storage conditions: +4 °C
Fetal Calf Serum (FCS)	Gibco	10270-106	Storage conditions: +4 °C
Fluoromount	Sigma	F4680	Mounting solution. Storage conditions: Room temperature
Fungizone - 50 mg	Bristol-Myers-Squibb	90129TB29	Anti-fungal reagent. Storage conditions: +4 °C
FX1 Input Straw with lock cap	Biolidics	CBB-F013005	Straw. Storage conditions: Room temperature
KovaSlide	Dutscher	50126	Chambered slide. Storage conditions: Room temperature
PanCK monoclonal antibody (clone AE1/AE3) Alexa Fluor 488	ThermoFisher	53-9003-80	Storage conditions: +4 °C
Paraformaldehyde 16%	ThermoFisher	11490570	Fixation solution. Storage conditions: +4 °C
PD-L1 monoclonal antibody (clone 29E2A3) - Phycoerythrin	BioLegend	BLE329706	Storage conditions: +4 °C
Petri Dish	Dutscher	632180	Storage conditions: Room temperature
Phosphate Buffered Saline (PBS)	Ozyme	BE17-512F	Storage conditions: +4 °C
Phosphate Buffered Saline Ultra Pure Grade 1x – 1 L	1st Base Laboratory	BUF-2040-1X1L	Storage conditions: Room temperature
Pluronic F-68 10%	Gibco	24040-032	Anti-binding solution. Storage conditions: Room temperature
Polylysine slides	ThermoFisher	J2800AMNZ	Storage conditions: Room temperature
Polypropylene Conical Tube 50 mL	Falcon	352098	Storage conditions: Room temperature
RBC Lysis Buffer – 100 mL	G Biosciences	786-649	Storage conditions: +4 °C
RBC Lysis Buffer – 250 mL	G Biosciences	786-650	Storage conditions: +4 °C
Resuspension Buffer (RSB)	Biolidics	CBB-F016003	Storage conditions: +4 °C
Shandon Cytopsin4 centrifuge	ThermoFisher	A78300003	Dedicated centrifuge. Storage conditions: Room temperature
Silicon Isolator	Grace bio-Labs	664270	Storage conditions: Room temperature
Sterile Deionized Water – 100 mL	1st Base Laboratory	CUS-4100-100ml	Storage conditions: Room temperature
Straight Fluorescent microscope Axio Imager D1	Zeiss		Storage conditions: Room temperature
Surgical Sterile Bag	SPS Laboratoires	98ULT01240	Storage conditions: Room temperature
Syringe BD Discardit II 20 mL sterile	BD Biosciences	300296	Storage conditions: Room temperature
Syringe Filter 0.22 µm 33 mm sterile	ClearLine	51732	Storage conditions: Room temperature
Zen lite 2.3 Lite Software	Zeiss		Microscope associated software