To determine the origin of cells forming the organoids, cells dissociated from tissues as mentioned above were stained with anti-E-cadherin (cat no. AF748; R&D Systems, Minnesota, United States) or anti-NGFR (cat no. sc-13577; Santa Cruz Biotechnology, Dallas, TX, USA) and their secondary antibody, Alexa Fluor 488–labeled donkey anti-goat IgG (cat no. A-11055; Invitrogen) or goat anti-mouse IgG (cat no. A-32723; Invitrogen). For direct labeling, FITC-conjugated anti-CD44 (cat no. 555478; BD Biosciences, Heidelberg, Germany), PE-conjugated anti-ITGA6 (cat no. 12-0495-83; BioLegend, San Diego, CA, USA), PE/Cyanine7-conjugated anti-NGFR Antibody (cat no. 345110; BioLegend) and APC-conjugated anti-EpCAM (cat no. 130-113-260; Miltenyi Biotec, Bergisch Gladbach, Germany) antibodies were used (Table S2 ). They were individually sorted using an S3e Cell Sorter (Bio-Rad, Hercules, CA, USA) and cultured in Matrigel with TeM for 15 days.
Fitc conjugated anti cd44
Manufactured by BD
Sourced in United States
FITC-conjugated anti-CD44 is a monoclonal antibody that binds to the CD44 cell surface glycoprotein. CD44 is involved in cell-cell interactions, cell adhesion, and migration. The FITC (fluorescein isothiocyanate) conjugation allows for the detection of CD44-expressing cells using fluorescence techniques.
Automatically generated - may contain errors
Lab products found in correlation
Fitc conjugated anti cd90, by BD (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Anti e cadherin, by R&D Systems (1 mentions)
Alexa fluor 488 labeled donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
S3e cell sorter, by Bio-Rad (1 mentions)
Goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti cd133, by Miltenyi Biotec (1 mentions)
Fitc conjugated anti cd44, by Miltenyi Biotec (1 mentions)
Pe conjugated anti cd24, by Miltenyi Biotec (1 mentions)
Perm wash buffer, by BD (1 mentions)
Fitc conjugated anti brdu monoclonal antibody, by BD (1 mentions)
Anti ifn γ, by BD (1 mentions)
Anti granzyme b, by BD (1 mentions)
Percp conjugated anti cd3, by BD (1 mentions)
Pe conjugated anti cd8, by BD (1 mentions)
Pe conjugated anti cd133, by Thermo Fisher Scientific (1 mentions)
Anti perforin, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Anti il 17, by BD (1 mentions)
10 protocols using fitc conjugated anti cd44
1
Organoid Formation from Dissociated Cells
2
Multiparametric analysis of stem cell markers
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2x106 cells were collected and stained with 20 μ of phycoerythrin (PE)-conjugated anti-CD24, anti-Sox2, anti-Oct3/4, and anti-Nanog antibodies, or fluorescein isothiocyanate (FITC)-conjugated anti-CD44 (BD Biosciences), or with PE-conjugated anti-CD133 (Miltenyi Biotech), or co-stained with FITC-conjugated anti-CD44 antibodies and PE-conjugated anti-CD24 or PE-conjugated anti-CD133. In the process of staining for Sox 2, Oct3/4 and Nanog, BD Perm/Wash buffer (BD biosciences) was used according to the manufacturer's instructions. PE- or FITC-positive cells were quantified on a LSRII flow cytometer (BD Biosciences), and up to 5x104 cells were counted per run.
For the bromodeoxyuridine (BrdU) incorporation assay, 10 μM BrdU was added to the cell suspension 2 hours before collection. Cells were then fixed with cold 70% ethanol, and labeled with a FITC-conjugated anti-BrdU monoclonal antibody according to the manufacturer's instructions (BD Biosciences). Propidium iodide was added before flow cytometric analysis. Detection of BrdU incorporation in DNA synthesizing cells was conducted by flow cytometry.
For the bromodeoxyuridine (BrdU) incorporation assay, 10 μM BrdU was added to the cell suspension 2 hours before collection. Cells were then fixed with cold 70% ethanol, and labeled with a FITC-conjugated anti-BrdU monoclonal antibody according to the manufacturer's instructions (BD Biosciences). Propidium iodide was added before flow cytometric analysis. Detection of BrdU incorporation in DNA synthesizing cells was conducted by flow cytometry.
3
Multicolor Flow Cytometry Analysis
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FITC-conjugated anti-CD44, PerCP-conjugated anti-CD3, PE-conjugated anti-CD8, FITC-conjugated anti-Granzyme A, anti-Granzyme B, anti-perforin and anti-IFN-γ were all purchased from BD Pharmingen. PE-conjugated anti-CD133 was purchased from eBiosciences. For cell surface staining, SMMC7721 and HepG2 cells were resuspended in 100μl staining buffer containing 10% FBS and put on ice for 20 min to block Fc receptors, then incubated with FITC-conjugated anti-CD44and PE-conjugated anti-CD133or isotype control for 30 min. The cells were then washed with 1ml ice-cold staining buffer for 2 times and centrifuged (300g) at 4°C for 5 min. The collected cells were suspended in 500μl staining buffer solution. PBMCs were incubated with SMMC7721 cells for 5 hours with brefeldin A at a final concentration of 10μg/ml, then collected and washed. As discussed above, PerCP-conjugated anti-CD3 and PE-conjugated anti-CD8 were used for surface staining. After that, cells were fixed and permeabilized, and then intracellular staining of FITC-conjugated anti-GranzymeA, Granzyme B, perforin and IFN-γ was performed. All samples after staining were evaluated using BD FACS Canto II with Diva software and analyzed using Flowjo 7.6.5.
4
CD24 Cell Depletion and Analysis
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CD24 expressing cells were depleted from A549 cell cultures using magnetic cell separation columns and CD24 antibody conjugated magnetic beads (Miltenyi Biotech) according to manufacturer’s instructions. Depletion was verified using flow cytometry as stated above. Antibody staining to determine CD44 and CD24 expression was performed using FITC conjugated anti-CD44 and Alexa 647 conjugated anti-CD24 from BD. Cells were stained and quantified by flow cytometry. Dead cells were identified and excluded using 4′,6-diamidino-2-phenylindole (DAPI) staining. Analysis was done in BD FACS Diva software.
5
Lymphocyte Isolation from Tissues
6
Characterizing EPO-Treated Bone Marrow Stem Cells
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BMSCs were trypsinized after incubation with or without EPO (500 IU/ml) for 48 h and washed three times with phosphate-buffered saline (PBS). Cell surface markers were examined by immunostaining with the following antibodies: phycoerythrin (PE)-conjugated anti-CD45 (BD Biosciences, San Jose, CA), fluorescein isothiocyanate (FITC)-conjugated anti-CD44 (BD Biosciences), and FITC-conjugated anti-CD90 (BD Biosciences) antibody. The labeled cells were analyzed using a flow cytometer (BD LSRFortessa™, Piscataway, NJ). Osteogenic and adipogenic differentiation potential of the cells in the two groups was examined according to the manufacturer’s protocol (Cyagen, China).
7
Multiparametric Flow Cytometry Immunophenotyping
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The cells were harvested as mentioned above and resuspended in PBS containing 1% FBS along with 0.5 mM EDTA. The cell suspension was incubated with specific antibodies for 1 h at 4°C. The following monoclonal antibodies were used: FITC (fluorescein isothiocyanate)-conjugated anti-CD 13 (ID Labs, # IDAC1071), FITC-conjugated anti-CD31 (Immunostep, # 31F-100T), FITC-conjugated anti-CD44 (BD Biosciences, # 560977), FITC-conjugated anti-CD 49b (BD Biosciences, # 555498), PE (phycoerythrin)-conjugated anti-CD 45 (BD Biosciences, # 555483) and PE-conjugated anti-CD146 (Biocytex, # 5050-PE100T). Thereafter, the cells were washed with PBS and fixed in 2% PFA. Analysis was performed on at least 10,000 events per sample using a FACS caliber flow cytometer (BD Biosciences). The acquisition was performed using CELLQUEST software (BD Biosciences) and analyzed using FCS-express software (DeNovo).
8
Characterizing Adipose-Derived Mesenchymal Stem Cells
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To characterize the MSCs, the expression of known surface proteins from AT-hMSCs was assessed by flow cytometry using a fluorescence-activated cell sorter (FACS Vantage; BD BioSciences, San Diego, CA). The data were analyzed using Cell Quest Pro software (BD BioSciences). AT-hMSCs and DHCs were harvested and stained with FITC-conjugated anti-CD90 (1∶100; Milternyi Biotec, Auburn, CA) or FITC-conjugated anti-CD44 (1∶1000; BD BioSciences) antibodies at 4°C for 10 min. A minimum of 10,000 events were recorded and analyzed.
9
Multi-Color Flow Cytometry for Cell Characterization
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The following commercially available antibodies were used for multi-color flow cytometry: BV510-conjugated anti-CD3, PE-conjugated anti-CD107a, APC-conjugated anti-ICAM1, PE-conjugated anti-EpCAM, PE-conjugated anti-CD13, FITC-conjugated anti-CD44, FITC-conjugated anti-CD90, PE-conjugated anti-CD133 (BD Biosciences, San Jose, CA, USA), APC-conjugated anti-CD56 (Miltenyi Biotec), APC-conjugated anti-MHC-1, APC-conjugated anti-ULBP-1, APC-conjugated anti-ULBP-2/5/6, APC-conjugated anti-ULBP-3, APC-conjugated anti-TRAIL-R1 (R&D Systems, Minneapolis, MN, USA), APC-conjugated anti-CEACAM1, APC-conjugated anti-HLA-G, and APC-conjugated anti-MICA/B (Biolegend, San Diego, CA, USA). Dead cells were excluded using the LIVE/DEAD red fluorescent reactive dye (Invitrogen). For some experiments, surface marker–stained cells were permeabilized using a Foxp3 Staining Buffer Kit (eBioscience) and further stained for PE-conjugated anti-aldehyde dehydrogenases (ALDH) (Sino Biological, PA, USA). Multi-color flow cytometry was performed using the Canto II instrument (BD Biosciences), and data were analyzed using the FlowJo software (TreeStar, Ashland, OR, USA).
10
Flow Cytometry Characterization of ASCs
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To confirm the ASC-progenitor phenotype, flow cytometry was performed after 3 days following the protocol in the previous subsection. Briefly, after tissue digestion ASCs (8 × 10 4 ) were washed, resuspended in 1% BSA in PBS, and incubated with FITC-conjugated anti-CD29, APC-conjugated anti-CD31, FITC-conjugated anti-CD44, FITCconjugated anti-CD45, and PE-conjugated anti-CD90 (BD Bioscience, San Jose, CA,USA), respectively, for 1 h at 4°C. Corresponding isotype-matched antibodies were used as controls. FACS analysis was performed using an Accuri C6 cytometer (BD, Franklin Lakes, NJ, USA).
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