The epidermis was separated from the dermis using 0.8% trypsin (50 min at 37 °C), minced and filtered through 70-μm and 40-μm cell strainers. Single-cell suspensions were subsequently incubated with primary antibodies in 5% FCS in PBS for 45 min at 4 °C. FACS analysis was performed using a FACSCanto II cytometers equipped with FACSDiva Software (BD). The following antibodies were used: CD49f-FITC (BD 555735; 1:500), CD49f-eFluor450 (eBioscience; 1:300), CD34 (eBioscience clone RAM34; 1:100). Cell viability was assessed by 7AAD (eBioscience; 1:50). Data were analysed using FlowJo 7.6 software.
Ram34
Manufactured by Thermo Fisher Scientific
Sourced in United States
The RAM34 is a versatile laboratory equipment designed for general-purpose laboratory applications. It features a compact and durable construction to withstand frequent use in laboratory environments. The core function of the RAM34 is to provide reliable and consistent performance for a variety of laboratory tasks.
Automatically generated - may contain errors
Lab products found in correlation
Cd49f fitc, by BD (1 mentions)
Facscanto 2 cytometer, by BD (1 mentions)
7 aad, by Thermo Fisher Scientific (1 mentions)
Facsdiva software, by BD (1 mentions)
Sa2 8, by Thermo Fisher Scientific (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Moflo xdp, by Beckman Coulter (1 mentions)
Pe rat anti mouse cd31, by BD (1 mentions)
Pe rat anti mouse cd45, by BD (1 mentions)
Rat anti mouse cd34 fitc, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by BD (1 mentions)
7 aad, by BD (1 mentions)
Mec13, by BD (1 mentions)
Ab37305, by Abcam (1 mentions)
Streptavidin peroxidase, by Vector Laboratories (1 mentions)
M7165, by Agilent Technologies (1 mentions)
Human cd44h 2c5, by R&D Systems (1 mentions)
Ab18207, by Abcam (1 mentions)
U7033, by Agilent Technologies (1 mentions)
Invitrogen goat antirabbit igg alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
8 protocols using ram34
1
Isolation and Characterization of Epidermal Cells
2
Characterization of Adipose-Derived Stem Cells
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ASCs were characterized via quantification of surface marker expression. The cells were dislodged by trypsin-ethylenediaminetetraacetic acid (EDTA) and stained with antibodies known to be expressed by MSCs (CD44: eBioscience clone IM7, San Diego, CA, USA; CD29: eBioscience clone HMb1-1) and hematopoietic cells (CD34: eBioscience clone RAM34; CD14: eBioscience clone Sa2-8) for 30 minutes at 4°C. Unstained cells served as a negative control. Data were acquired on a BD FACS Canto Flow Cytometer (BD Biosciences) and analyzed with FlowJo software (Treestar). The cells were gated based on their forward and side scatter properties to exclude debris and doublets. A fuller characterization of these cells as well as a colony-forming unit fibrolasts (CFU-F) assay was performed and reported in a separate publication to verify ASC characteristics [32 (link)]. Cells of interest stained positive for both F480 and CD11b (general macrophage markers).
3
Skeletal Muscle Stem Cell Isolation
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For isolation of SCs by FACS sorting, skeletal muscles from hind limbs were prepared similarly as for flow cytometry analysis, resuspended in PBS + 2% FBS, and then incubated with the following antibodies for 30 min on ice: rat anti-mouse α7integrin-APC (1:15, 334,908, R&D Systems), rat anti-mouse CD34-FITC (1:30, RAM34, eBioscience), rat anti-mouse CD45-PE (1:30, 30-F11, BD Biosciences), rat anti-mouse CD31-PE (1:30, MEC13.3, BD Biosciences), and rat anti-mouse Sca-1-PE-Cy7 (1:30, D7, eBioscience). After incubation, cells were washed, filtered through a 40-μm cell strainer, and resuspended in PBS + 2% FBS with Hoechst 33342 (10 μg/ml) and 7-AAD (1:40, BD Biosciences). Cells were sorted with MoFlo XDP (Beckman Coulter) cell sorter.
4
Immunohistochemical Analysis of Blood Vessels
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Example 7
Tissue sections were process as described (Sukhdeo, K. et al. Proc Natl Acad Sci USA 104, 7516-21 (2007)). Sections were incubated with primary antibodies (5 μg/ml) or the corresponding IgG fraction of preimmune serum overnight at 4° C. in blocking solution (3% BSA/PBS). BCL9 (ab37305, Abcam), mouse CD34 (RAM34, eBiosciences), human CD34 (M7165, Dako) and human CD44H (2C5, R&D Systems) antibodies were employed. Blood vessel formation in the CRC and MM models was evaluated using anti-mouse CD34 and anti-human CD34 antibodies, respectively, and the corresponding biotinylated antibodies coupled to streptavidin peroxidase (Vector). The number of blood vessels was determined by counting the mean number of independent blood vessels in 5 randomly selected fields at 50× magnification as highlighted by CD34 staining (brown color). See results in
5
Penile Tissue Fixation and Immunostaining
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Penile tissues were harvested and fixed overnight at 4°C in 4% (w/v) PFA dissolved in PBS The detailed protocols were previously described.1 , 2 , 31 , 33 For primary antibody staining, the following antibodies were utilized: anti‐CD34 (1/200, RAM34, eBioscience™), anti‐ACTA2 (1/1000, U7033, DAKO), and anti‐β‐Ⅲ‐tubulin (1/200, ab18207, Abcam). For secondary antibody reaction, the following antibodies were utilized: Invitrogen goat antirabbit IgG Alexa Fluor 488 and Invitrogen goat antimouse IgG Alexa Fluor 488 (1/200, Thermo Fisher Scientific).
6
Isolation of Mouse Hematopoietic Stem Cells
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(c-kit+Sca1+Lin−) LT-HSCs or ST-HSCs were sorted, as described previously [36] (link). In brief, we isolated bone marrow cells from 8- to 10-week-old C57BL/6 mice and stained them with antibodies for CD34 (RAM34, eBiosciences, San Diego, CA), Sca-1 (E13-161.7, BD Biosciences Pharmingen, San Jose, CA), c-kit (2B8, BD Biosciences Pharmingen), and a lineage marker (Lineage Detection Kit, Miltenyi Biotec Inc., Bergisch Gladbach, Germany). Subsequently, we analyzed the stained cells using a MoFlo XDP cell sorter system (Beckman Coulter, Fullerton, CA).
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(c-kit+Sca1+Lin−) LT-HSCs or ST-HSCs were sorted, as described previously [36] (link). In brief, we isolated bone marrow cells from 8- to 10-week-old C57BL/6 mice and stained them with antibodies for CD34 (RAM34, eBiosciences, San Diego, CA), Sca-1 (E13-161.7, BD Biosciences Pharmingen, San Jose, CA), c-kit (2B8, BD Biosciences Pharmingen), and a lineage marker (Lineage Detection Kit, Miltenyi Biotec Inc., Bergisch Gladbach, Germany). Subsequently, we analyzed the stained cells using a MoFlo XDP cell sorter system (Beckman Coulter, Fullerton, CA).
7
HSC Immunophenotyping and FACS Sorting
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BM cells were incubated with antibodies for 30 min on ice. BM cells were stained for HSCs and progenitors using antibodies CD4 (GK1.5), CD8 (53-6.7), CD11b (M1/70), Gr-1 (RB6-8C5), B220 (RA3-6B2), and TER119 (TER119); CD16/32 (93), CD41 (MW Reg 30), and CD105 (MJ7/18); and cKit (2B8), Sca-1 (D7), CD150 (TC15-12F12.1), CD48 (HM48-1), and CD34 (RAM34; eBioscience). When comparing untreated to pI:C/LPS-treated HSCs, Sca-1 was excluded from the gating because of its strong up-regulation. After 5-FU treatment, c-Kit was excluded from the HSC gating scheme, as its expression on HSCs is lost during 5-FU recovery. Cells were analyzed using an LSRII or LSR Fortessa flow cytometer (BD). Data were analyzed using FlowJo software (Tree Star). For FACS sorting, FACSAria I, II, and III cell sorters were used (BD).
8
Multiparametric Immunohistochemistry Analysis
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Sections 5 μm in thickness were de-paraffinised and re-hydrated. A pretreatment method using heat-induced epitope retrieval was used, and the nonspecific binding was blocked with a protein blocker (DakoCytomation, Trappe, France) for 30 min at room temperature (RT). The sections were then incubated with the following primary antibodies against Ki67 (ab15580; Abcam), EpCam (clone G8.8; Biolegend), α-SMA (ab21027; Abcam), CD34 (RAM34, eBioscience), GP38 (MA615113; Thermo Fisher), CD24 (ab64064; Abcam), Lysozyme (ab108508; Abcam), Muc2 (Thermo Fisher) and ZO-1 (Invitrogen) antibodies.
The co-labelling with antibodies from the same species was performed using Opal™ Multiplex IHC kits (Akoya). After incubation with the primary antibodies, the slides were washed in PBS-T three times and probed with appropriated fluorescence-conjugated secondary antibodies for 1 h at room temperature. After three washings in PBS, the cell nuclei were counterstained by Vectashield mounting medium with DAPI (Vector).
The co-labelling with antibodies from the same species was performed using Opal™ Multiplex IHC kits (Akoya). After incubation with the primary antibodies, the slides were washed in PBS-T three times and probed with appropriated fluorescence-conjugated secondary antibodies for 1 h at room temperature. After three washings in PBS, the cell nuclei were counterstained by Vectashield mounting medium with DAPI (Vector).
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