Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

Summary

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the "myovascular unit".

Abstract

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional "myovascular unit" (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.

This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Introduction

Via bloodstream, cells and organs are supplied with oxygen, substrates and other necessary molecules. This interchange takes place in capillaries, the smallest vessels. Capillaries are formed by an inner endothelial cell (EC) layer whose integrity remains a prerequisite to successful regulation of muscle homeostasis between the intravascular and interstitial space. To ensure a selective transition of soluble factors and cells, EC constitute a monolayer interconnected by tight and adherens junctions ¹. Besides its role as barrier for nutrients or metabolic products, EC regulate the recruitment of leukocytes in inflammatory processes. Inflammation or tissue damage leads to an up-regulation of adhesion molecules on the EC surface and production of chemokines facilitating leukocyte attachment and transmigration into the target tissue ². Consequently, EC are critically involved in the regulation of inflammatory processes such as the defense against pathogens or tissue repair.

A dysfunction of EC is directly associated with vascular diseases, chronic kidney failure, venous thrombosis severe pathogen infections. Furthermore, EC are virtually always involved in organ-specific autoimmunity such as diabetes mellitus or multiple sclerosis ³. The barrier function between blood stream and organs is therefore controlled by a concerted interplay of different cell types. In the skeletal muscle microvascular endothelial cells (MMEC) together with muscle cells, fibroblasts and pericytes form a functional unit, the "myovascular unit" (MVU). Therefore, a dysfunction of the MVU might play a critical role in the pathophysiology of myopathies. However, a deeper understanding of these regulatory mechanisms is still missing and currently precludes the identification of new, urgently needed, therapeutic targets in myopathies.

To investigate the complex physiological and pathophysiological mechanisms, animal models are commonly used. However, in vitro models offer the advantage to focus on the subject of interest by excluding a variety of confounding factors. To investigate processes in vitro it is necessary to isolate pure and viable primary cells. In contrast to cell lines, primary cells isolated from transgenic animals enable to investigate the consequences of genetic modifications in vitro.

Here, a method to isolate primary murine MMEC is described by using mechanical and enzymatic dissociation followed by magnetic activated cell sorting techniques (MCS) for purification. For this purpose, magnetic beads against specific surface markers are used. Platelet endothelial cell adhesion molecule-1 (PECAM1, CD31) is mainly expressed on EC and can be used to enrich this cell type. To warrant high cell purity, cells of hematopoietic origin are excluded by a negative selection for protein tyrosine phosphatase receptor type C (PTPRC, CD45). Further, quality controls, cultivation of primary murine MMEC, potential applications and limitations as well as special considerations are presented.

Protocol

All animal experiments were approved by the local authorities and conducted according to the German animal welfare act (84-02.05.20.13.097).

1. General Remarks on Animal Experiments

1. Perform all mouse experiments in accordance with the guidelines of the respective institutional animal care and use committee.
2. Keep mice under standardized conditions and according to international guidelines such as the Federation for Laboratory Animal Science Associations (FELASA).
   NOTE: In general this isolation technique can be used for mice independent of age, gender or genetic background. To obtain a sufficient cell number, 4–10 week old males are preferred, because biological properties can vary with age and gender.

2. Preparation of Solutions, Media and Coating

1. Prepare digestion solution (DS) by mixing 2.2 mL of Dulbecco´s Modified Eagle Medium (DMEM) with 200 μL Collagenase-Dispase and 45 μL Desoxyribonuclease (DNase).
2. Prepare 500 mL of Endothelial cell medium (ECM) by mixing 450 mL of DMEM with 50 mL FCS (approximately 10%), 0.25 mL basic fibroblast growth factor bFGF (20 μg/mL; approximately 0.05%) and 5 mL penicillin-streptomycin (approximately 1%). Perform sterile filtration in a glass tube (diameter of filter pores: 0.2 μm). Afterwards store the solution at 4 °C.
3. Coat cell culture plates as described below.
   1. For cultivation of freshly isolated primary murine MEC cover the whole surface with 1 mL per well of a 6 well cell culture plate with a gelatin based speed coating solution (see Table of Materials) according to manufacturer’s instructions for 3–5 min at room temperature (RT).
   2. Aspirate and discard coating solution. Rinse each well with 2 mL sterile phosphate buffered saline (PBS), pH 7.1–7.5 in two consecutive washing steps.
   3. Aspirate and discard PBS. Fill up wells with 1 mL volume of ECM.

3. Isolation of Primary Murine Muscle Microvascular Endothelial Cells (MMEC)

1. Euthanize one adult 4–12 weeks old, male mouse by cervical dislocation without any anaesthesia. The aim is to quickly separate the spinal cord from the brain so as to provide the animal with a fast and painless death.
2. Sever the extremities.
   1. Use a surgical sharp/blunt (cutting edge 42 mm, straight) and sharp/sharp (cutting edge 23 mm, straight) scissor, a straight (serrated, 2 mm x 1.25 mm x 12 cm) and a curved (serrated, 1.3 mm x 1 mm x 13 cm) forceps.
   2. Disinfect all surgical instruments with 70% ethanol.
   3. Position the animal on its back, moisten the legs with 70% ethanol. Sever the whole leg by cutting at the hip joint with the sharp/blunt surgical scissor. Place the extremities in a closed cell culture dish (35 mm x 10 mm).
      NOTE: From now on perform every step under a sterile laminar flow hood and work with sterilized instruments.
3. Isolate muscle tissue (preferentially use the musculus quadriceps femoris or musculus triceps surae from both legs).
   1. Cut the skin open from the hip to the toe-tip by using a sharp/sharp scissor and curved forceps. Hold the toe or footpad with the straight forceps. Peel off the skin with the curved forceps from the toe to the hip.
   2. Isolate the musculus quadriceps femoris by cutting the tendon from the knee and sever the muscle along the femur to the hip. Isolate the musculus triceps surae by severing the Achilles tendon. Hereafter, cut along the tibia to the popliteal fossa and remove the muscle.
      NOTE: If large vessels (A. femoralis, A. tibialis anterior, A. tibialis posterior, A. fibularis) are visible in the isolated muscles, remove vessels to avoid contamination by macrovascular endothelium.
   3. To remove large vessels, hold the part of muscle not containing large vessels with a curved forceps and cut it off next to the vessel. For this protocol, 1 g or less muscle tissue equal to both quadriceps femoris and triceps surae is recommended.
   4. Add 2,445 µL DS (from step 2.1) to a cell culture dish (35 mm x 10 mm) and determine the weight.
   5. Transfer all muscle pieces to this cell culture dish containing the 2445 µL DS and determine the weight. The difference of both measured values provides the dry weight of muscle tissue which must not exceed 1 g.
   6. Cut the whole muscle tissue into small pieces (≤2 mm cubes, about 100 pieces) by using the sharp/sharp scissor.
4. Dissociate the muscle tissue.
   NOTE: This step takes about 120 min.
   1. Store muscle/DS suspension at 37 °C (incubator with 5% CO₂) for 1.5 h. Mix the suspension carefully every 20 min for about 5 min using a 1 mL insulin syringe.
   2. Transfer suspension to a 70 μm nylon cell strainer placed on a 50 mL tube and collect the flow through. Wash the cell strainer with 8 mL of DMEM and collect the flow through.
   3. Discard cell strainer and centrifuge suspension for 10 min at 300 x g at 20 °C. Carefully remove supernatant.
      CAUTION: pellet is easy to lose.
   4. Resuspend cell pellet in 1 mL of an Ammonium-Chloride-Potassium (ACK) lysing buffer (see enclosed Table of Materials) for the lysis of red blood cells and incubate for 30 s at RT. Add 9 mL DMEM + 10% FCS to stop the reaction and transfer cell suspension to a 15 mL tube.
5. Deplete CD45⁺ cells following the CD45 microbeads manufacturer’s instructions. For all following steps of CD45⁺ depletion use MCS buffer (see enclosed Table of Materials).
   NOTE: This step takes about 60 min.
   1. Determine cell numbers using a Neubauer cell counting chamber (expect about 5 x 10⁶ cells per g muscle tissue).
   2. Centrifuge suspension at 300 x g for 10 min at 4 °C. Take off supernatant completely and resuspend cell pellet in 90 µL MCS buffer (per 10⁷ cells or less). Add 10 μL of CD45 microbeads (per 10⁷ cells or less).
   3. Mix cell suspension and incubate for 15 min in the fridge at 4–8 °C.
   4. Add 1mL MCS buffer (per 10⁷ cells or less). Centrifuge at 300 x g for 10 min at 4°C. Remove the supernatant completely and resuspend cells in 500 μL MCS buffer.
   5. For magnetic cell separation use a large magnetic column (LMC) with a reservoir volume of 8 mL and a capacity of up to 2 x 10⁹ total cells. Position the LMC in the magnetic field of the separator.
   6. Rinse with 3 mL of MCS buffer into the reservoir of the LMC. After rinsing, place a 15 mL conical tube below the column to collect flow through. Apply the whole cell suspension (500 µL) onto the column and let it completely flow through.
   7. Wash the column three times by adding 3 mL of MCS buffer into the reservoir and wait until the column´s reservoir is empty before performing the next washing step.
   8. Collect the unlabeled cells passing the column use for further separation steps (representing the CD45^- fraction). Labeled cells (representing the CD45⁺ fraction) accumulated in the column can be discarded.
6. Accumulate CD31⁺ cells following CD31 microbeads manufacturer’s instructions. For all following steps of CD31⁺ accumulation use MCS buffer.
   NOTE: This step takes about 60 min.
   1. Determine cell number using a Neubauer cell counting chamber (expect about 4 x 10⁶ cells per g muscle tissue).
   2. Centrifuge obtained unlabeled cell suspension from step 3.5 for 10 min at 300 x g at 4°C.
   3. Remove the supernatant completely. Resuspend the pellet in 90 μL MCS buffer and add 10 μL of CD31 microbeads (per 10⁷ total cells or less). Mix the whole suspension and incubate for 15 min in the fridge at 4–8 °C.
   4. Add 1mL MCS buffer and centrifuge at 300 x g for 10 min at 4 °C. Take off the supernatant and resuspend cells in 500 µL of MCS buffer.
   5. For the magnetic cell separation use a medium magnetic column (MMC) with a reservoir volume of 3.5 mL and a capacity of up to 2 x 10⁸ total cells.
   6. Position the MMC in the magnetic field of the separator. Rinse the MMC with 500 µL of MCS buffer. After rinsing, place a 15 mL conical tube below the column to collect flow through.
   7. Apply the whole cell suspension (500 µL) onto the column and let it completely flow through.
   8. Wash the column three times by adding 500 µL of MCS buffer and wait until the column’s reservoir is empty before performing the next washing step. Unlabeled cells passing the column represent the CD45^- CD31^- fraction. Store them for further quality controls.
   9. Remove column from the separator and place it on a suitable collection tube (15 mL tube). Pipette 2 mL MCS buffer onto the column. Immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column. This CD45^- CD31⁺ fraction represents the enriched primary murine EC.
   10. Centrifuge cell suspension for 5 min at 350 x g at 20 °C. Remove supernatant completely and resuspend cells (expect around 0.6 x 10⁶ cells per g muscle tissue) in 1 mL ECM (see step 2.2.). Transfer cells (0.5–0.7 x 10⁶ cells) to a single well of a coated 6-well culture plate (from step 2.3) containing 1 mL of ECM.
7. For cultivation, incubate cells at 37 °C and 5% CO₂ in a sterile incubator. Refresh the ECM every two to three days.

4. Primary Murine MMEC Purification

1. Perform a second cycle of CD31⁺ accumulation, following manufacturer’s instructions (CD31 microbeads mouse protocol; see step 3.6.).
   1. Detach cells with Trypsin/EDTA solution when they are at 80–90% confluence (usually after 7 days). For this, rinse each well with 2 mL sterile PBS, in two consecutive washing steps. Use 800 μL Trypsin/EDTA solution per 6-well and incubate at 37 °C and 5% CO₂ in a sterile incubator for 3–5 min. Stop enzymatic activity by using 1200 μL DMEM containing a minimum of 10% FCS.
   2. Centrifuge the cell suspension for 5 min at 350 x g at 20 °C.
   3. Perform the second CD31-MCS step to increase the purity (according to the CD31 microbeads manufacturer´s instructions; see 3.6.).
   4. Observe cell confluence via bright field or standard phase-contrast microscopy using a
      20x magnification and 0.35 lens numerical aperture. See Figure 1.
      NOTE: Cells can be used for respective experiments or kept in culture by passaging. Use cells for experiments promptly in lower passages. It is not recommended to use cells after passage
      8–10.

5. Quality Control

1. Perform flow cytometry of primary murine MMEC after the second CD31-MCS-step.
   1. Prepare a FACS tube by adding 1 mL flow cytometry (FC) buffer (see enclosed Table of Materials).
   2. Detach cells with Trypsin/EDTA solution when they are at 100% confluence (usually after 14 days). For this, rinse each well with 2 mL sterile PBS, in two consecutive washing steps. Use 800 μL Trypsin/EDTA solution per 6-well and incubate at 37 °C and 5% CO₂ in a sterile incubator for 3–5 min. Stop enzymatic activity by using 1200 μL DMEM containing 10% FCS.
   3. Determine cell number using a Neubauer cell counting chamber and add a minimum of 1 x 10⁵ cells to the prepared FACS tube.
   4. Centrifuge cell suspension for 10 min at 300 x g at 4 °C. Perform two washing steps by removing the supernatant, resuspending cells in 1 mL FC buffer and centrifuging for 10 min at 300 x g at 4 °C.
   5. Prepare a staining mix by adding anti-mouse CD31-FITC antibody (1:100) and anti-mouse CD45-PE (1:100) with FC buffer.
   6. Resuspend cells in 100 μL of staining mix in the FACS tubes. Incubate for 30 min at 4 °C. Add 1 mL of FC buffer. Centrifuge cell suspension for 5 min at 300 x g at 4 °C. Carefully remove supernatant and resuspend cells in 200 μL of FC buffer.
   7. Measure cells in a flow cytometer following the gating strategy is shown in Figure. 2A.
2. Alternatively perform immunofluorescence staining of primary murine MMEC after the second CD31-MCS-step.
   1. Coat the coverslips.
      1. Transfer a sterile 13 mm diameter round glass coverslip into a well of a 24 well plate. Coat coverslip by adding 300 µL of speed coating solution per well. Incubate for 3–5 min at room temperature (RT).
      2. Aspirate and discard coating solution. Rinse each well with 2 mL sterile PBS in two consecutive washing steps. Aspirate and discard PBS.
      3. Seed 1 x 10⁵ cells in 1 mL ECM on the coated coverslip and incubate at 37 °C and 5% CO₂ in a sterile incubator until cells are 99% confluent.
   2. Perform fixation and immunofluorescence staining.
      1. Remove the ECM medium of cultivated cells from 5.2.1.3.
      2. Fix cultured cells by adding 300 µL 4% paraformaldehyde (PFA) with pH of 7.4 per well, for 10 min at 4°C.
         CAUTION: PFA is toxic and must be handled with care.
      3. Perform 3 washing steps by adding 1 mL PBS per well and remove supernatant after 5 min at RT.
      4. Prepare a blocking solution containing 5% BSA, 0.2% Triton-X and 1% goat serum in PBS.
      5. Add 300 µL of blocking solution to each well and incubate for 1 h at RT.
      6. Remove the supernatant and add 200 µL per well of an anti-PECAM-1 (CD31) antibody (1:200) in 5% BSA, 1% goat serum in PBS and incubate at 4 °C overnight.
      7. Perform 3 washing steps by adding 1 mL PBS per well and removing supernatant after 5 min at RT in each step.
      8. Prepare a secondary antibody solution containing Cy3-conjugated anti-rat IgG antibody (1:500) in 1% BSA and PBS. Add 300 µL per well of the secondary antibody solution and incubate for 1 h at RT in the dark.
      9. Perform 3 washing steps by adding 1 mL PBS per well and removing supernatant after 5 min at RT in each step.
   3. Take out the round glass coverslips carefully with forceps and transfer it onto a microscopy slide. Add an appropriate amount of mounting medium containing DAPI on the cell layer and carefully put a cover glass on it (see enclosed Table of Materials).
   4. Observe cells with an appropriate fluorescence microscope equipped with a fluorescence light source (120 W) and two filter sets: An excitation/emission filter set with 550/25 nm and 605/70 nm wavelengths to detect Cy3 540/562 nm.
   5. Use a second filter set with an excitation/emission wavelength of 365/50 nm and 445/50 nm to detect DAPI 350/440 nm. Use a magnification of 40x and 80% intensity of 14.5 mW/cm² for DAPI with an acquisition duration of 30 ms. For detection of Cy3 use 80% of 67.5 mW/cm² with an acquisition duration of 60 ms.
3. Perform quantitative PCR.
   1. Isolation of mRNA.
      1. Follow standard procedures using an acid guanidinium thiocyanate-phenol (AGTP) reagent to isolate mRNA. Use a minimum of 0.5 x 10⁶ cells from CD45^- CD31^- (see step 3.6.8), CD45^- CD31⁺ (see step 3.6.9.) fractions and cultivated primary murine MMEC (4.1.3)
      2. Centrifuge cell suspension for 10 min at 300 x g at 20 °C. Take off the supernatant completely. Resuspend the cell pellet in 500 µL of AGTP reagent and transfer cell suspension into a 2 mL reaction tube. Incubate for 5 min at RT.
      3. Add 100 µL of chloroform and shake vigorously for 15 s. Incubate for 3 min at RT.
      4. Centrifuge suspension for 15 min at 12,000 x g at 4°C.
      5. Take off the upper aqueous phase (containing RNA) carefully and transfer into a 1.5 mL reaction tube. Add 250 µL of isopropanol and incubate for 10 min at RT.
      6. Perform a centrifugation step at 12,000 x g for 10 min at 4 °C.
      7. Take off the supernatant and add 1 mL of 75% ethanol carefully. Centrifuge at 7,500 x g for 5 min at 4 °C.
         NOTE: Pellet is easy to lose.
      8. Remove the supernatant completely. Let the pellet dry on air for 5–10 min.
         NOTE: ethanol needs to be removed but do not dry too much.
      9. Dissolve pellet in 30 µL diethylpyrocarbonate treated water containing in and heat for 10 min at 55 °C.
         NOTE: RNA concentration and purity can be measured by a spectral-photometer.
   2. Perform cDNA synthesis (reverse transcriptase PCR).
      1. Prepare a mastermix containing: 10 µL of PCR buffer (5x), 2,5 µL desoxyribonucleosid-triphosphate (dNTP (10 mM)), 0.5 µL random mixture of single-stranded primer (0.2 µg/ µL), 1 µL of RNase inhibitor (40 U/ µL, 0.4 reverse transcriptase (200 U/µL) and 5.6 µL of diethylpyrocarbonat treated water (see enclosed Table of Materials).
      2. Dilute 500 ng RNA in 30 µL diethylpyrocarbonate treated water in 0.2 mL PCR tubes and add the whole 20 µL mastermix.
      3. Use a PCR cycler performing following steps: 10 min 25 °C, 30 min 50 °C, 5 min 85 °C and hold at 4 °C.
4. Perform Quantitative PCR (qPCR).
   NOTE: Perform qPCR of samples in duplicates or triplicates.
5. Use primer with two different fluorescence dyes. Primer with an absorption of 495 nm and an emission of 517 nm for the gene of interest.
6. For detection of the satellite cell marker gene expression, use primers for paired box protein 7 (Pax7) and M-cadherin (Cdh15) (see enclosed Table of Materials).
7. For detection of the endothelial cell marker gene expression, use primers for claudin-5 (Cld5), occludin (Ocln) and zonula occludens-1 (Tjp1 or ZO1) (see enclosed Table of Materials).
8. As reference gene for 18s rRNA, use a primer with absorption of 538 nm and an emission of 554 nm wavelength.
9. Prepare a qPCR mastermix for each gene of interest with the respective primer. Mastermix contains: 10µl qPCR buffer (2x), 1 µL of Primer for gene of interest (10 µM), 1 µL of Primer for 18s rRNA and 4 µL of nuclease free water.
10. Add 16 µL of the mastermix to a single well of a 96-well reaction plate. Add 4 µL of cDNA (obtained from step 5.3.2.) per well containing the mastermix.
11. Use a real-time PCR cycler performing following steps: holding stage with 50 °C for 2 min and 95 °C for 10 min. Cycling stage with 40 cycles of 95 °C 15 s and 60 °C for 1 min.

Results

One day after isolation, primary murine MMEC and residual other cells form conglomerates and adhere to the bottom of culture dishes (Figure 1A day 1). From day 7 on, flat and elongated cells can be observed. However, contamination of other, mostly spheroid cells, is still visible (Figure 1A day 7). Thus, another cycle of CD31 positive selection via MCS is required. Hereafter, primary murine MMEC proliferate to a density ...

Discussion

Microvascular endothelial cells provide barrier functions in all tissues and their dysfunction results in disease of the associated organs ³. Moreover, organ-specific studies of microvascular EC could pave the way for new therapeutic strategies. Therefore, a deeper understanding of microvascular EC function under physiological and pathophysiological conditions is of great scientific interest. Modulation of leukocyte/endothelium interaction is successfully used to treat multiple sclerosis patients ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA	Thermo Fisher	25200-056	ready to use
ACK buffer			150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA in water at a pH of 7.3
Anti-mouse CD31-FITC (clone MEC13.3)	Biolegend	102506	Isotype control: FITC Rat IgG2a, κ Isotype Ctrl
Anti-mouse CD45-PE (clone 30-F11)	Biolegend	103106	Isotype control: PE Rat IgG2b, κ Isotype Ctrl
bFGF	Peprotech	100-18B	Basic fibroblast growth factor
BSA	Sigma Aldrich	A4503
CD31 MicroBeads mouse	Miltenyi Biotec	130-097-418
CD45 MicroBeads mouse	Miltenyi Biotec	130-052-301
Collagenase-Dispase	Roche	10269638001	Collagenase from V. alginolyticus, Dispase from B. polymyxa
Corning Costar TC-Treated Multiple 6-Well Plates	Corning	3516
Cy3-conjugated anti-rat IgG antibody	dianova	712-166-153
DAPI (ProLong Gold antifade reagent with DAPI)	Thermo Fisher	P36935
Desoxyribonuclease	Sigma Aldrich	D4513	Deoxyribonuclease I from bovine pancreas
Diethylpyrocarbonat treated water	Thermo Fisher	AM9916
DMEM, containing Glutamin Supplement and pyruvate	Thermo Fisher	31966-021	warm up to 37 °C before use
dNTP Mix (10 mM)	Thermo Fisher	R0192	1 mL
EDTA	Sigma Aldrich	E5134
FACS tubes	Sarstedt	551,579
Falcon 70 μm Cell Strainer	Corning	352350
FC buffer			0.1% BSA, 0.2% NaN3, 2 mM EDTA
Fetal calf serum	Sigma Aldrich	F6178	Fetal calf serum
Fixable Viability Dye eFluor780	Thermo Fisher	65-0865-14
Forceps (serrated, straight, 12 cm)	Fine Science Tools	11002-12
Forceps (serrated, straight, 12 cm)	Fine Science Tools	11009-13
Insulin syringe 100 Solo 1 mL (Omnifix)	Braun	9161708V
large magnetiv columns (LS columns)	Miltenyi Biotec	130-042-401	for CD45-MACS-step
MCS buffer			0.5% BSA, 2 mM EDTA in PBS at a pH of 7.2
Medium magnetic column (MS column)	Miltenyi Biotec	130-042-201	for CD31-MACS-step
Nuclease free water	Thermo Fisher	R0581
PBS	Sigma Aldrich		Phosphate buffered saline, ready to use
PCR buffer (5x)	Thermo Fisher	EP0742	in a kit with the reverse transcriptase
Pecam1 rat α-mouse	SantaCruz	Sc-52713	100 µg/mL
Penicillin-Streptomycin	Sigma Aldrich	P4333
primary murine muscle cells	celprogen	66066-01
Primer Cdh15 (M-Cadherin)	Thermo Fisher	Mm00483191_m1	FAM labeled
Primer Cldn5 (claudin-5)	Thermo Fisher	Mm00727012_s1	FAM labeled
Primer Ocln (occludin)	Thermo Fisher	Mm00500912_m1	FAM labeled
Primer Pax-7	Thermo Fisher	Mm01354484_m1	FAM labeled
Primer Tjp-1 (Zonula occludens 1)	Thermo Fisher	Mm00493699_m1	FAM labeled
Primer 18s rRNA (Eukaryotic endogenous control)	Thermo Fisher	4310893E	VIC labeled
qPCR buffer (Maxima Probe/ROX qPCR Master Mix (2X)	Thermo Fisher	K0231	2 x 1,25 mL; for 200 reactions each
Random mixture of single-stranded primer	Thermo Fisher	SO142	Random Hexamer Primer
Reverse Transcriptase (200 U/μL) + PCR buffer (5x)	Thermo Fisher	EP0742
Rnase Inhibitor (40 U/μL)	Thermo Fisher	EO0381
Scissor (cutting edge 23 mm, sharp/sharp)	Fine Science Tools	14088-10
Scissor (cutting edge 42 mm, sharp/blunt)	Fine Science Tools	14001-13
Speed Coating solution	PeloBiotech	PB-LU-000-0002-00