As soon as a frozen MSC batch has passed the final release, any bag of this batch can be thawed at the LTCG and infused into a patient included in a clinical trial. Previously, cells were thawed and diluted in PBS (1:0.75 MSC:PBS) (Miltenyi Biotec, Bergisch Gladbach, Germany) as described previously [9 (link)]. The same procedure is applied now, except that we use saline instead of PBS, and the dilution ratio is slightly different (1:0.5 MSC:NaCl). Cell count and viability are assessed by the Nucleocounter method. The cell product is then transferred in an appropriately labeled sterile transfer bag and transported to the hematology ward for infusion into the patient.
Phosphate buffered saline (pbs)
Manufactured by Miltenyi Biotec
Sourced in Germany
PBS (Phosphate-Buffered Saline) is a common laboratory buffer solution used to maintain the pH and osmotic balance of biological samples. It is a balanced salt solution composed of sodium chloride, potassium chloride, sodium phosphate, and potassium phosphate. PBS is designed to mimic the ionic composition of human body fluids, making it suitable for a wide range of applications in cell and molecular biology.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Miltenyi Biotec (3 mentions)
Automacs, by Miltenyi Biotec (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
Ovomucoid, by Worthington (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Miltenyi Biotec (1 mentions)
Dnase 1, by Worthington (1 mentions)
Erythrocyte lysis buffer, by Merck Group (1 mentions)
Dmem 10 fbs, by Thermo Fisher Scientific (1 mentions)
Collagenase, by Worthington (1 mentions)
Collagenase type 1, by Worthington (1 mentions)
Cd73 pe, by Miltenyi Biotec (1 mentions)
Hla dr pc5, by Beckman Coulter (1 mentions)
Cd45 pc7, by BD (1 mentions)
Macsquant analyzer, by Miltenyi Biotec (1 mentions)
Cd166 pe, by BD (1 mentions)
Collagenase 2, by Worthington (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Ficoll gradient, by GE Healthcare (1 mentions)
Rbc lysis buffer, by Thermo Fisher Scientific (1 mentions)
Macsquant system, by Miltenyi Biotec (1 mentions)
11 protocols using phosphate buffered saline (pbs)
1
MSC Infusion Protocol for Clinical Trials
2
Kupffer Cell Isolation from Injured Liver
3
Purification and Culture of Mouse Retinal Ganglion Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse RGCs were purified using a magnetic-bead separation method that has been previously described.18 (link) Briefly, P0 to P5 mouse retinas from CD1 mice were dissected in Mg2+/Ca2+-free Hanks' balanced salt solution (HBSS). Retinas were then digested for 5 minutes at 37°C in HBSS containing 20 U/mL papain and 0.005% DNase I (Worthington Biochemicals, Lakewood, NJ, USA). Digestion was neutralized with ovomucoid and 0.005% DNase I (Worthington Biochemicals) and then the retina was triturated with a pipette. Dissociated cells were treated with rabbit anti-mouse Thy1.2 antibody conjugated to micrometal beads (130-049-101; Miltenyi Biotech, Auburn, CA, USA) for 15 minutes at room temperature in elution buffer (phosphate-buffered saline with 0.5% bovine serum albumin and 2 mM EDTA; Miltenyi Biotech). RGCs were then purified from the cell suspensions using a metal column in the presence and absence of a magnetic field. Purified RGCs were quantified and then diluted to 0.5 × 106 RGCs/mL in medium (see above). Cells (1 mL) were then plated onto an electrotaxis chamber (see below) and placed in incubator at 37°C overnight for 12 hours.
4
Adipose-Derived Stem Cell Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
First, 100 cc of fat was removed from the thigh area of each patient. The tissue was then washed with phosphate-buffered saline (PBS) (Miltenyi Biotec, Cologne, Germany) to remove red blood cells and leukocytes. The adipose tissue was digested with collagenase type I (Worthington Biochemical Corp, Lakewood, USA) for 20 min at 37 °C to produce a collagenase solution with a concentration of 0.1%. Enzyme digestion was prevented by washing with DMEM 10% FBS (Invitrogen, Carlsbad, USA), and floating and lysed fat cells were discarded. SVF cells were pelleted by centrifugation at 500g for 10 min. The pellet was resuspended in PBS, and an erythrocyte lysis buffer (Sigma-Aldrich Corp, St. Louis, USA) was added and incubated at 37 °C for 10 min. This cell suspension was centrifuged (500g, 5 min), and SVF cells were counted using an automatic cell counter.
The viability of isolated SVF cells was evaluated in the laboratory using an automatic cell counter. Flow cytometry was performed to analyze the surface marker expression of SVF cells. The data analyses were conducted using Partec—CyFlow ML. Data analysis was carried out using FloMax® software.
The viability of isolated SVF cells was evaluated in the laboratory using an automatic cell counter. Flow cytometry was performed to analyze the surface marker expression of SVF cells. The data analyses were conducted using Partec—CyFlow ML. Data analysis was carried out using FloMax® software.
5
IL-23 Induced Skin Inflammation
6
Multiparametric Analysis of Mesenchymal Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested after detachment with TrypLE Select, washed in PBS (Miltenyi Biotec, Bergisch Gladbach, Germany) and incubated for 30 min with the following monoclonal antibodies: CD105-FITC (Ancell corporation, Bayport, NY, USA), CD73-PE (Miltenyi Biotec, Bergisch Gladbach, Germany), CD146-PC5 (Beckman Coulter, Analis, Suarlée, Belgium), CD166-PE (BD Biosciences, Erembodegem, Belgium), CD45-PC7 (BD Biosciences, Erembodegem, Belgium), HLA-ABC- PC5 (BioLegend, San Diego, CA, USA), HLA-DR-PC5 (Beckman Coulter, Analis, Suarlée, Belgium), CD47-APC (Miltenyi Biotec, Bergisch Gladbach, Germany) and CD200-PC7 (BD). After washing with PBS, the cells were fixed with 8% formaldehyde. Data were acquired and analyzed on a MacsQuant Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany).
7
Isolation of Immune Cells from Blood and Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immune cells were isolated from blood and tissue as previously described 2 , 3 (link), 9 . Peripheral blood (∼100 μl) was collected in tubes with ethylenediaminetetraacetic acid (EDTA) (BD Biosciences, San Jose, California). Erythrocytes were lysed with RBC lysis buffer (eBioscience, Thermo Fisher Scientific, Waltham, Massachusetts), and the remaining cells were washed with phosphate-buffered saline (PBS) and fixed with 1% paraformaldehyde. Mediastinal lymph nodes (LNs) were flushed with magnetic-activated cell sorting buffer containing bovine serum albumin, EDTA, and PBS (Miltenyi Biotec, Bergisch Gladbach, Germany), and cells were subsequently centrifuged at 500 g for 5 min at 4°C. After resuspension in PBS, cells were fixed with 1% paraformaldehyde. Whole hearts were flushed with PBS, minced into small pieces using a single-edged blade, and digested in gently agitated RPMI medium with 1 mg/ml collagenase-2 (Worthington Biochemical, Lakewood, New Jersey), 1 mg/ml trypsin (Invitrogen, Carlsbad, California), and 10 μg/ml DNase I at 37°C for 45 min. Cell suspensions were filtered through a 70-μm cell strainer and incubated in 2 mmol/l EDTA/PBS for 5 min at 37°C. Isolates were then centrifuged on a Ficoll gradient (GE Healthcare, Little Chalfont, United Kingdom) (2000 g for 20 min), and mononuclear cells were collected, washed with PBS, and fixed with 1% paraformaldehyde.
8
Cell Cycle Analysis Using PI
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For cell cycle analysis, propidium iodide (PI) was applied for nuclear staining as described by C. Riccardi and I. Nicoletti [43]. 2.5 × 104 cells were washed twice with PBS (Miltenyi) (7 minutes/4 °C/550 g). Cells were resuspended in 25 μL staining solution and incubated in the dark for 1 h/4 °C prior to flow cytometry (Beckman Coulter).
9
Quiescent CD34+ Cell Treatments
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quiescent CD34+cells were subjected to treatment immediately after isolation, whereas cycling cells were expanded for 5 days prior to treatment.
One hundred millimolars and 1M stock solutions of Etoposide (TCI EUROPE NV) and MNU (MedChemExpress) were prepared in DMSO (Wak-Chemie Medical GmbH), respectively. Etoposide treatment was conducted for 24 h with following concentrations: 1.5 µM/5 µM/10 µM. MNU treatment was conducted for 1 h with following concentrations for qPCR: 1 mM/3 mM/5 mM and 0.1 mM/0.3 mM/0.5 mM for all other experiments. After treatment, cells were washed with PBS (Miltenyi) prior to further cultivation or subsequent analysis (Supplementary Fig. S2 ).
One hundred millimolars and 1M stock solutions of Etoposide (TCI EUROPE NV) and MNU (MedChemExpress) were prepared in DMSO (Wak-Chemie Medical GmbH), respectively. Etoposide treatment was conducted for 24 h with following concentrations: 1.5 µM/5 µM/10 µM. MNU treatment was conducted for 1 h with following concentrations for qPCR: 1 mM/3 mM/5 mM and 0.1 mM/0.3 mM/0.5 mM for all other experiments. After treatment, cells were washed with PBS (Miltenyi) prior to further cultivation or subsequent analysis (
10
Immunophenotyping of Cell Populations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell surface marker staining was performed by direct immunofluorescence with conjugated monoclonal antibodies listed in Table S4 . Briefly, a sample of 1 × 105 cells was incubated with conjugated antibodies in 50 μl of phosphate‐buffered saline (PBS) (Lonza, USA) containing 1% (v/v) bovine serum albumin (BSA) (Grifols, Spain) (PBS‐BSA) for 30 min at room temperature (RT) with continuous mixing in the dark. Cells were washed twice with PBS‐BSA and then analysed on a MACSQuant flow cytometer using MACSQuantify software (Miltenyi Biotech, Germany). For RhAG staining, a sample of 5 × 105 cells was incubated with the monoclonal antibody LA1818 (kindly provided by Prof. Ellen van der Schoot, Sanquin, Netherlands) for 30 min at RT, and then labelled with FITC‐conjugated goat anti‐mouse IgG and IgM (Becton Dickinson) for 30 min at RT in the dark. Results were analysed on a MACSQuant system (Miltenyi Biotech). Cell viability was assessed by 7‐AAD staining.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!