CD31 MicroBead Kit
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1. Figure 1 Epidermis dermis and hypodermis form a three tiered structure To access HDMECs within the dermal layer middle the epidermis can be removed by peeling the top layer from the dermis and hypodermis 140 001 341 04 2 Protocols Purification of CD31 cells isolated from Rhesus monkey Macaca mulatta tissue 2 Protocols The following section describes the extraction of CD31 endothelial cells from two different tissue sources for the subsequent purification with CD31 MicroBeads Dermal microvascular endothelial cells can be extracted from human foreskin biopsies section 2 1 while venous endothelial cells can be obtained from the umbilical cord section 2 2 2 1 Preparation of human dermal microvascular endothelial cells HDMECs 2 1 1 Principle of HDMEC purification This protocol describes the purification of CD31 HDMECs from foreskin tissue Firstly neutral protease treatment of the biopsy using Dispase II enables the separation and removal of the epidermal layer from the dermis Once separated the dermis itself is digested by Collagenase Type la to leave a cell suspension containing HDMECs which are then cultivated for a minimum of 24 hours Cultivation facilitates an increased purity and yield of CD31 HDMECs after CD31 MicroBead selection by removal of non adherent CD31 cells After treatment of the cultured cells with trypsin HDMECs in a single cell suspension are purified using the CD31 MicroBead Ki2. 75 cm cell culture flasks Lab equipment Centrifuge Laminar flow hood biohazard containment hood Microscope hemocytometer Water bath 37 C 140 001 341 04 2 Protocols Fill a 20 mL syringe with HepesBSS 6 mM EDTA and rinse the vein via the feeding needle until no remnants of blood are visible in the eluate A Note CD31 is expressed on certain populations of blood cells as well as platelets Thorough washing ensures minimal contamination after CD31 MicroBead Kit purification Insert a second feeding needle into the vein at the other end of the cord and fix with forceps Check for a continuous flow from this end by rinsing the vein once more with HepesBSS 6 mM EDTA Fill a second syringe with 5 mL of Trypsin EDTA Invitrogen pre warmed to 37 C and replace the first syringe without the formation of air bubbles Inject 4 5 mL of the Trypsin EDTA Invitrogen Before injecting the last 0 5 mL seal the opposite end with a Double Dead Ender Cap to prevent collapse of the vein Suspend the cord in a U shape in the 500 mL beaker with PBS pre warmed to 37 C Incubate for 20 min at 37 C Place the cord on a soft surface e g tissue paper and carefully massage for 5 min with two fingers Put 2 mL of FBS into a 50 mL conical tube Fill a syringe with 8mL of HepesBSS 6mM EDTA and attach without letting air bubbles in 140 001 341 04 2 Protocols Reagents PBS without Ca or Mg
3. CD31 MicroBead Kit 3 2 Separation of HUVECs Separation of CD31 HUVECs prepared from a human umbilical cord using CD31 MicroBeads and a MidiMACS Separator with an LS Column The cells are fluorescently stained with CD31 APC 130 092 652 and CD45 PE 130 080 201 a marker to indicate leukocyte contamination CD45 CD31 double positive cells in the positive fraction reflect the insufficient removal of blood from the cord Cell debris and dead cells were excluded from the analysis based on scatter signals and PI fluorescence CD31 cells Before separation CD31 cells er CD45 PE CD45 PE CD45 PE CD31 APC CD31 APC CD31 APC 140 001 341 04 All protocols and data sheets are available at www miltenyibiotec com Warnings Reagents contain sodium azide Under acidic conditions sodium azide yields hydrazoic acid which is extremely toxic Azide compounds should be diluted with running water before discarding These precautions are recommended to avoid deposits in plumbing where explosive conditions may develop Warranty The products sold hereunder are warranted only to be free from defects in workmanship and material at the time of delivery to the customer Miltenyi Biotec GmbH makes no warranty or representation either expressed or implied with respect to the fitness of a product for a particular purpose There are no warranties expressed or implied which extend beyond the technical specifications of the products Milteny
4. biopsy when cutting with a scalpel as grip is achieved between the underside and the dish in contrast to that of the smooth epidermal side 140 001 341 04 2 Protocols Identify the inner and outer sides of the foreskin the inside is a lighter color than the outside Separate the two sides by carefully cutting between them with a round bladed scalpel A Note Foreskin biopsies can be cut easier and cleaner by using short measured steps and with a back and forth rocking motion A clean cut will later facilitate a simpler separation of the epidermis from the dermis step 9 Carefully subdivide both pieces into smaller pieces of approximately 4 5 mm Place all the pieces into the pre prepared petri dish containing Dispase II with the epidermal side facing downwards A Note Orienting the tissue with the epidermal side facing downwards is very important in order to achieve an adequate separation of dermis from epidermis Incubate for 12 24 h at 4 C After the incubation prepare a 15 mL conical tube with 10 mL of HepesBSS Separate the dermis from the epidermis carefully peel back the epidermal layer of all the pieces using sterile tweezers epidermis can be differentiated easily by its darker coloration and wafer like appearance see diagram on page 6 Collect the dermal layers in the 10 mL of HepesBSS Let the pieces settle to the bottom before aspirating the supernatant A Note A small volume of supernatant r
5. tissue After removing blood from the outside of the umbilical cord both ends as well as damaged tissue caused by the clamping of the ends should be removed Next all blood within the umbilical vein is thoroughly but carefully washed out before injecting trypsin into the umbilical cord vein in order to release the endothelial cells After an incubation period trypsinized HUVECs are then collected by eluting the contents of the cord vein and are directly centrifuged to remove the trypsin before proceeding with the isolation of CD31 cells using the CD31 MicroBead Kit see section 2 3 Briefly CD31 HUVECs are immunolabeled with CD31 MicroBeads before being loaded onto a column placed in the magnetic field of a MACS Separator The magnetically labeled CD31 cells are retained on the column The unlabeled cells run through and this cell fraction is depleted of CD31 cells After removal of the column from the magnetic field the magnetically retained CD31 cells can be eluted as the positively selected cell fraction and directly taken into culture or analyzed for purity by flow cytometry 140 001 341 04 2 Protocols 2 2 2 Reagent and instrument requirements Materials 500 mL glass beaker Sterile tweezers Sterile scalpel Sterile 20 mL syringe Sterile Gavage Feeding Needles Fine Science Tools 18061 20 Sterile clamps e g Carl Roth N141 1 Double Dead Ender Cap male Qosina 65802 Sterile 50 mL conical tubes T 75
6. transendothelial migration of leukocytes endothelial cells play a key role in the inhibition of inflammation thrombosis vascular smooth muscle proliferation and the promotion of vasodilation by its release of nitric oxide NOY The functional integrity of the endothelium is an important area of cardiovascular research especially its dysfunction in the formation of atherosclerotic lesions The isolation of endothelial cells from foreskin biopsies and umbilical cord vein tissue using CD31 MicroBeads permits the in vitro study of highly pure endothelial cell cultures 1 2 Product applications Purification of CD31 HDMECs isolated from human foreskin tissue Purification of CD31 HUVECs isolated from human umbilical cord tissue Enrichment of HDMECs or HUVECs in fibroblast contaminated cell cultures Purification of other CD31 cell types such as CD4 RTEs after prior untouched enrichment of T cells using the Naive CD4 T Cell Isolation Kit 130 091 894 140 001 341 04 2 Protocols being loaded onto a column placed in the magnetic field of a MACS Separator The magnetically labeled CD31 cells are retained within the column The unlabeled cells run through this cell fraction is thus depleted of CD31 cells After removing the column from the magnetic field the magnetically retained CD31 cells can be eluted as the positively selected cell fraction and directly taken into culture or analyzed for purity by flow cytometry
7. HepesBSS 6 mM EDTA Trypsin EDTA Invitrogen 25300 054 Trypan blue Fetal bovine serum FBS 2 2 3 Experimental procedures A As with the isolation of HDMECs from foreskin umbilical cords should be thoroughly scrutinized for sites of damage which must be re moved before the endothelial cell isolation procedures are started Intact umbilical cords should be at least 12 cm long Perform all steps within a laminar flow hood 1 To remove injured ends of the cord or the damage caused by clamping cut neatly on the interior side of the injury with a scalpel and at a distance of at least 1 cm A Note To obtain a neat straight cut grip the cord with a pair of sterile tweezers and lead the scalpel along the inside line of the tweezers cutting carefully At one end insert a feeding needle carefully into the vein without damaging the surrounding tissue Fix in place with clamp A Note The umbilical vein can be easily recognized as it is the vessel with the largest diameter 140 001 341 04 2 Protocols Remove cap from opposite end carefully flush the cord with Hepes EDTA and collect all effluent in the conical tube containing the FBS The eluate should be cloudy with flakes of cellular material Centrifuge eluate at 300xg for 5 min at room temperature Resuspend cells well in 1 mL of PBS and count viable cells by Trypan blue exclusion in a hemocytometer Proceed directly to purification of CD31 cells using the CD31
8. MicroBead Kit see 2 3 CD31 MicroBead Kit purification 2 3 CD31 MicroBead Kit purification A For the purification of HDMECs EndoGMMV should be used throughout the procedure Similarly EndoGM should be used for HUVEC purification 2 3 1 Reagent and instrument requirements LS Column MidiMACS Separator Optional Fluorochrome conjugated antibody for flow cytometric analysis for example CD31 PE 130 092 653 CD31 APC 130 092 652 CD45 FITC 130 080 202 CD34 PE 130 081 002 CD34 APC 130 090 954 Optional Propidium iodide PI or 7 AAD for flow cytometric exclusion of dead cells 140 001 341 04 2 Protocols Optional Pre Separation Filters 130 041 407 to remove cell clumps 2 3 2 Magnetic labeling A Volumes given are for up to 1x10 cells When working with fewer than 1x10 cells use the same volumes as indicated When working with higher cell numbers scale up all reagent volumes and total volumes accordingly e g for 2x10 total cells use twice the volume described 1 Centrifuge cells at 300xg for 3min Aspirate supernatant completely Resuspend cells to a maximum concentration of 1x10 cells per 60 uL of medium Add 20 uL of FcR Blocking Reagent per 1x10 cells Vortex briefly then add 20 uL of CD31 MicroBeads to the mixture Incubate 15 min at 4 C Add 1 mL of medium per 1x10 and centrifuge cells at 300xg for 3 min Resuspend the cell pellet in 1 mL of
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10. aining stabilizer and 0 05 sodium azide Cross reactivity CD31 MicroBeads are reported to react with rhesus monkey Macaca mulatta cells Storage Store protected from light at 2 8 C Do not freeze The expiration date is indicated on the vial label 1 1 Background information The CD31 MicroBead Kit has been developed for the isolation of human dermal microvascular endothelial cells HDMECs from foreskin biopsies as well as human umbilical vein endothelial cells HUVECs CD31 also known as PECAM 1 platelet endothelial cell adhesion molecule 1 is a single chain 130 kDa transmembrane glycoprotein that belongs to the immunoglobulin superfamily and mediates cell to cell adhesion CD31 is found constitutively expressed on the surface of microvascular lymphatic umbilical cord and pulmonary capillary endothelial cells as well as on platelets monocytes polymorphonuclear cells and discrete populations 140 001 341 04 140 001 341 04 1 Description of lymphocytes including CD4 RTEs recent thymic emigrants CD31 is central to the transendothelial migration of leukocytes Cell to cell interactions via CD31 occur homophilically with CD38 or with avB3 integrin as ligands CD31 is also involved in angiogenesis Endothelial cells form the layer of thin flat cells that line the inside of blood vessels forming a barrier between blood in the vessel lumen and the rest of the vessel wall In addition to the regulation of
11. ation CD31 cells CD31 cells Relative cell number Relative cell number Relative cell number i _ 5 i pee CD31 APC CD31 APC CD31 APC 140 001 341 04 3 Examples of separations using the CD31 MicroBead Kit Figure 2 Cultured HDMECs after extraction from foreskin tissue and purification using the CD31 MicroBead Kit 140 001 341 04 4 Related products 4 Related products 130 092 653 130 092 652 130 081 001 130 081 002 130 090 954 130 080 202 130 080 201 130 050 601 130 090 753 DeLisser H M et al 1994 Molecular and functional aspects of PECAM 1 CD31 Immunol Today 15 490 495 CD31 PE human CD31 APC human CD34 FITC human CD34 PE human CD34 APC human CD45 FITC human CD45 PE human Anti Fibroblast MicroBeads human MACSmix Tube Rotator Kimmig S et al 2002 Two subsets of naive T helper cells with distinct T cell receptor excision circle content in human adult peripheral blood J Exp Med 195 789 794 Muller W A et al 1993 PECAM 1 is required for transendothelial migration of leukocytes J Exp Med 178 449 460 Bird I N et al 1999 Homophilic PECAM 1 CD31 interactions prevent endothelial cell apoptosis but do not support cell spreading or migration J Cell Sci 112 1989 1997 Behrendt D and Ganz P 2002 Endothelial function From vascular biology to clinical applications Am J Cardiol 90 40L 48L 140 001 341 04 3 Examples of separations using the
12. ed regi ons be observed then Braunol treatment is mandatory Perform all steps within a laminar flow hood Prepare 5x50 mL conical tubes containing 10 mL Braunol solution 15 mL PBS 15 mL PBS 15 mL HepesBSS 0 05 sodium thiosulfate 20 mL HepesBSS Immerse the tissue biopsy into Braunol solution using sterile tweezers Incubate the tube at room temperature for 10 min on an orbital shaker or MACSmix Tube Rotator Transfer the biopsy to 15 mL of PBS and incubate for a further 5 min with agitation Transfer the biopsy to the tube containing 15 mL of HepesBSS 0 05 sodium thiosulfate using sterile tweezers Incubate for 5 min at room temperature on an orbital shaker or MACSmix Tube Rotator 140 001 341 04 2 Protocols Pre treatment with Braunol solution Orbital shaker or MACSmix Tube Rotator 130 090 753 Braunol solution povidone iodine B Braun Melsungen 3864235 Sodium thiosulfate Sigma 7026 diluted to 0 05 in Hepes buffered saline solution HepesBSS e g PromoCell C 40020 Phosphate buffered saline PBS without Ca or Mg Reagents for enzymatic digestion and HDMEC extraction Dispase II Roche Diagnostics 295825 Collagenase Type la Sigma C2674 diluted to 0 25 in 2 mM CaCl HepesBSS HepesBSS Endothelial Cell Growth Medium MV EndoGMMV PromoCell C 22020 Reagents for HDMEC culture step Endothelial Cell Growth Medium MV EndoGMMV PromoCe
13. emaining in the tube will not disturb the subsequent collagenase digest 140 001 341 04 2 Protocols 2 1 5 Harvesting of cultivated cells A For the isolation of CD31 HDMECs cells are first trypsinized and then counted before proceeding to the CD31 MicroBead purification of HDMECs Cell confluency should optimally be 60 80 at time of harvesting resulting in a total yield of approximately 3 5x10 cells per foreskin biopsy of which around 3 5x10 are CD31 1 Remove culture supernatant and wash cells once with PBS to remove residual medium Add 100 uL of Trypsin EDTA PromoCell per cm of cultured cells and incubate at room temperature for several minutes A Note Incubation conditions for trypsinization may vary according to manufacturer of trypsin Always follow manufacturer s guidelines if different to those above Check under a microscope that the cells are completely dissociated If not gently tap flask on the bench or prolong incubation time A Note Avoid over trypsinization of cells Once cells are detached add 100 uL of Trypsin Neutralizing Solution per cm of cultured cells Resuspend cells and transfer them to a 15 mL conical tube Centrifuge cells at 300xg for 3 min Resuspend cells thoroughly in 1 2 mL of medium Count cells using a hemocytometer Proceed with 2 3 CD31 MicroBead Kit purification 140 001 341 04 2 Protocols Apply 5 mL of pre warmed 37 C Collagenase Type 1a solution and clo
14. further information refer to our website www miltenyibiotec com For technical questions please contact your local subsidiary or distributor vO LvVE LOO OrL Technical Support Team Germany E mail macstec miltenyibiotec de Phone 49 2204 8306 830 Contents 1 Description 1 1 Background information 1 2 Product applications Protocols 2 1 Preparation of human dermal microvascular endothelial cells HDMECs 2 1 1 Principle of HDMEC purification 2A 2 Experimental overview 2 1 3 Reagent and instrument requirements 2 1 4 Experimental procedures 2 1 4 1 Optional Pre treatment with Braunol solution 2 1 4 2 Extraction of HDMECs from foreskin tissue 2 1 5 Harvesting of cultivated cells Preparation of human umbilical vein endothelial cells HUVECs 2 251 Principle of HUVEC purification 2 2 2 Reagent and instrument requirements 2 263 Experimental procedures CD31 MicroBead Kit purification 2 3 1 Reagent and instrument requirements Madd Magnetic labeling 2 3 3 Magnetic separation Examples of separations using the CD31 MicroBead Kit 3 1 Separation of HDMECs 3 2 Separation of HUVECs Related products References 1 Description 1 Description 2 mL CD31 MicroBeads MicroBeads conjugated to monoclonal anti human CD31 antibody isotype mouse IgGl Components 2 mL FcR Blocking Reagent Human IgG Capacity For 10 total cells up to 100 separations Product format CD31 MicroBeads are supplied as a suspension cont
15. i Biotec GmbH s liability is limited to either replacement of the products or refund of the purchase price Miltenyi Biotec GmbH is not liable for any property damage personal injury or economic loss caused by the product Unless otherwise specifically indicated Miltenyi Biotec products and services are for research use only and not for diagnostic or therapeutic use autoMACS and MACS are registered trademarks of Miltenyi Biotec GmbH MidiMACS MiniMACS OctoMACS QuadroMACS SuperMACS VarioMACS and MACSmix are trademarks of Miltenyi Biotec GmbH Braunol is a registered trademark of B Braun Melsungen AG Copyright 2010 Miltenyi Biotec GmbH All rights reserved 140 001 341 04
16. ll C 22020 PBS without Ca or Mg Trypsin EDTA PromoCell C 41010 Trypsin Neutralizing Solution PromoCell C 41100 8 Trypan blue 140 001 341 04 2 Protocols Wash once more for 5 min with agitation in a second tube of 15 mL PBS Store the biopsy in 20 mL of HepesBSS until further use A Note Keep storage time of biopsies to an absolute minimum as cell viability decreases rapidly over time resulting in a lower yield of HDMECs after CD31 MicroBead isolation Do not store biopsies overnight or for 1 day before proceeding to the extraction step 2 1 4 2 Extraction of HDMECs from foreskin tissue For an illustrated short protocol please visit www miltenyibiotec com protocols HDMECs should be extracted from foreskin biopsies that are as fresh as possible and not older than 1 day The quantity of HDMECs recovered is severely affected in biopsies older than 1 day to the degree of a 10 fold reduction in a 5 day old sample 1 Prepare a sterile petri dish 60 mm in diameter with 5 6 mL of Dispase II solution per foreskin biopsy being prepared Place the biopsy in a second petri dish 100 mm in diameter assess for inflammation and cut away infected or damaged areas with a sterile scalpel followed by treatment with Braunol solution see 2 1 4 1 Orientate the biopsy so that the epidermal side smooth surface is facing upwards A Note Orientation in this fashion is to facilitate the easier handling of the
17. medium Proceed to magnetic separation see 2 3 2 140 001 341 04 2 Protocols Cells should be seeded at a density of approximately 150 000 per T 75 flask Culture conditions 37 C 5 CO and gt 95 humidity A Note Should fibroblast contamination outgrow endothelial cells upon prolonged cultivation endothelial cells should be re purified using CD31 MicroBeads or alternatively fibroblasts can be removed using Anti Fibroblast MicroBeads 130 050 601 Magnetic separation with the autoMACS Separator A Refer to the autoMACS user manual for instructions on how to use the autoMACS Separator 1 Prepare and prime autoMACS Separator 2 Place tube containing the magnetically labeled cells in the autoMACS Separator For a standard separation choose one of the following separation programs Positive selection Possel Depletion Depletes A Note Program choice depends on the isolation strategy the strength of magnetic labeling and the frequency of magnetically labeled cells For details see autoMACS user manual section autoMACS Cell Separation Programs When using the program Possel collect positive fraction from outlet port pos1 This is the purified positive cell fraction When using the program Depletes collect unlabeled fraction from outlet port neg1 This is the negative cell fraction 140 001 341 04 2 Protocols 2 3 3 Magnetic separation Magnetic separation with LS Columns 1 Place an LS C
18. olumn in the magnetic field of a MidiMACS Separator Prepare column by rinsing with 3 mL of medium 2 3 Apply cell suspension onto the column 4 Collect unlabeled cells which pass through and wash column with 3x3 mL of medium Perform washing steps by adding medium three times Only add new medium when the column reservoir is empty Collect total effluent this is the unlabeled cell fraction Remove column from the separator and place it on a suitable collection tube Pipette 5 mL of medium onto the column Immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column Cells can be directly analyzed by flow cytometry for purity or taken into culture For HDMECs yield of CD31 cells depends upon the size and thickness of foreskin used Generally cells isolated from a single 4 cm biopsy should be cultured in one T 75 flask For HUVECs yield of CD31 cells will depend upon cord length and efficiency of enzyme digestion 140 001 341 04 3 Examples of separations using the CD31 MicroBead Kit 3 Examples of separations using the CD31 MicroBead Kit Separation of CD31 HDMECs from a preparation of dermal layer cells of human foreskin using CD31 MicroBeads and a MidiMACS Separator with an LS Column The cells are fluorescently stained with CD31 APC 130 092 652 Cell debris and dead cells were excluded from the analysis based on scatter signals and PI fluorescence HDMECs before separ
19. se the tube Incubate for 75 min maximum in a 37 C waterbath Shake vigorously every 30 min A Note Optional Seal the tube with Parafilm to ensure that there is no contamination or sample loss A Note After digestion the cell suspension should be slightly viscous though it is normal to still observe undigested parts of skin tissue Dilute the cell suspension with 10 mL of EndoGMMV and pass through a 70 um nylon filter which is placed over the opening of a 50 mL conical tube Centrifuge filtrate at 300xg for 3 5 min Aspirate supernatant Resuspend pellet carefully in 1 mL of EndoGMMV Place cells into a T 75 75 cm culture flask and add a further 20 mL of EndoGMMV Culture the cells at 37 C 5 CO and gt 95 humidity for a minimum of 24 h before purification with CD31 MicroBead Kit A Note Endothelial cells become identifiable after 24h in culture and prolonged culture will increase yield of CD31 cells after CD31 MicroBead Kit isolation However growth of fibroblasts will also occur simultaneously and care must be taken to monitor for their overgrowth Therefore it is advised not to culture cells for longer than 3 days Proceed to purification of CD31 cells using the CD31 MicroBead Kit see 2 3 140 001 341 04 2 Protocols 2 2 Preparation of human umbilical vein endothelial cells HUVECs 2 2 1 Principle of HUVEC purification This protocol describes the purification of CD31 HUVECs from umbilical cord
20. t Briefly CD31 HDMECs are immunolabeled with CD31 MicroBeads before 140 001 341 04 2 Protocols 2 1 2 Experimental overview Foreskin biopsy not older than 1 day 4 Optional Braunol treatment for samples with inflammation 4 Dispase II digestion overnight 4 C 4 Separation of dermis from epidermis 4 Collagenase Type la digestion 1 2 h 37 C 4 Filtration of cell suspension 70 um nylon mesh 4 Cultivation in EndoGMMV 24 h 4 Trypsination 4 HDMEC purification with the CD31 MicroBead Kit 140 001 341 04 2 Protocols 2 1 3 Reagent and instrument requirements Materials Sterile tweezers Sterile round bladed scalpels e g B Braun Melsungen 5518083 Sterile Petri dish 100 mm in diameter Sterile Petri dish 60 mm in diameter Sterile pipettes Sterile 15 mL conical tubes Sterile 50 mL conical tubes 70 um nylon cell filter T 75 75 cm cell culture flasks Lab equipment Centrifuge Laminar flow hood biohazard containment hood CO incubator 37 C with 5 CO in air and gt 95 humidity Microscope hemocytometer Water bath 37 C 140 001 341 04 2 Protocols 2 1 4 Experimental procedures 2 1 4 1 Optional Pre treatment with Braunol solution A A pre treatment step in order to disinfect the foreskin biopsy using Braunol solution is strongly recommended Also foreskin biopsies may contain inflamed regions upon assessment Should large inflam
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