Anti-CD19 CAR T cells were co-cultured with target cell lines with designated E:T ratios (1:1 and 5:1) for 4 hours (Table 1 ). CD19 positive Raji cells and CD19 negative K562 cells were used as positive and negative control cells respectively. Target cells were subsequently stained with PE-conjugated mouse anti-human CD19 antibody (BD Biosciences, San Jose, CA) and analyzed by FACSCanto flow cytometry. The target cells were suspended in RPMI-1640 containing 10% FBS, labeled with calcein AM (BD Biosciences, San Jose, CA), and plated onto 96-well flat-bottom plates (Costar, Corning, NY). UWC19 cells, suspended in RPMI-1640 containing 10% FBS, were then added at various E:T ratios and co-cultured with the target cells for 4 h. Following this, the cells were stained with propidium iodide (Sigma-Aldrich, St. Louis, MO), and the cytotoxicity was assessed by flow cytometry on a FACSCanto (Becton Dickinson) instrument enumerating the number of viable target cells (calcein AM-positive, propidium-iodide negative, and light scattering properties of viable cells).
Calcein am
Manufactured by BD
Sourced in United States, United Kingdom, Germany
Calcein AM is a cell-permeant dye that can be used to stain live cells. It is a non-fluorescent compound that becomes highly fluorescent upon intracellular esterase activity, producing a bright green fluorescence. This allows for the detection and quantification of viable cells in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Calcein am, by Thermo Fisher Scientific (15 mentions)
Matrigel, by BD (11 mentions)
Tcl buffer, by Qiagen (8 mentions)
Facsaria fusion special order system, by BD (8 mentions)
Axio observer z1, by Zeiss (7 mentions)
Matrigel matrix, by BD (6 mentions)
Cd45 vioblue direct antibody conjugate, by Miltenyi Biotec (5 mentions)
Propidium iodide, by Merck Group (5 mentions)
Propidium iodide, by BD (5 mentions)
Fluoroblok cell culture inserts, by BD (5 mentions)
96 well flat bottom plate, by Corning (4 mentions)
Facscanto, by BD (4 mentions)
Facscalibur, by BD (4 mentions)
Facsaria fusion, by BD (3 mentions)
Cell dissociation buffer, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Cd45 vioblue, by BD (2 mentions)
Pe conjugated mouse anti human cd19 antibody, by BD (2 mentions)
Cd45 vioblue, by Miltenyi Biotec (2 mentions)
Spark 10m multimode microplate reader, by Tecan (2 mentions)
148 protocols using calcein am
1
Cytotoxicity Assay for Anti-CD19 CAR T Cells
2
Cytotoxicity Assay for NK Cells
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The target cells were suspended in RPMI-1640 containing 10% FBS, labeled with calcein AM (BD Biosciences, San Jose, CA), and plated onto 96-well flat-bottom plates (Costar, Corning, NY). The NK cells, suspended in RPMI-1640 containing 10% FBS, were then added at various E:T ratios and co-cultured with the target cells for 4 h. Following this, the cells were stained with propidium iodide (Sigma-Aldrich, St. Louis, MO), and the cytotoxicity was assessed by flow cytometry on a FACSCalibur (Becton Dickinson) instrument enumerating the number of viable target cells (calcein AM-positive, propidium-iodide negative, and light scattering properties of viable cells) [3] (link), [26] (link).
3
Measuring NK Cell Cytotoxicity
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For measurements of NK cell cytotoxicity, the calcein-AM release assay was performed as previously described with minor changes.19 (link) K562 target cells were stained with calcein-AM [10 μM; BD Pharmingen] at 37°C in 5% CO2 for 30 min. calcein-AM-stained cells were thoroughly washed to remove excess dye. PBMCs and stained K562 cells were co-cultured in triplicate at an effector-to-target ratio [E:T] of 10:1 and 5:1. For maximum release [positive control], stained target cells were lysed with 10% Triton-X solution. Cells were incubated for 2 h at 37°C in 5% CO2. After the incubation period, the cell plate was gently centrifuged, and supernatant was carefully aspirated and placed into a black 96-well plate, and calcein-AM fluorescence was measured by a FLUOstar OPTIMA Microplate Reader [BMG LABTECH]. When NK cells lyse target cells, the fluorescent dye is released into the supernatant. Therefore, fluorescence is proportional to the target cell killing calculated as follows:
4
Single-Cell RNA-Seq of Tumor Cells
5
Purification of Viable Tumor Cells
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Dissociated tumour cells (from fresh primary tumours and PDXs) were resuspended in cold 1% bovine serum albumin in phosphate buffered saline (PBS-BSA1%). Cells were first stained first with CD45-Vioblue direct antibody conjugate (Miltenyi Biotec, 130–092–880) in PBS-BSA1% for 20 min at 4°C, washed and then costained with 1μM calcein AM (Life Technologies, C3100MP) and 0.33 μM TO-PRO-3 iodide (Life Technologies, T3605) in PBS-BSA1%. Sorting was performed with FACSAria Fusion (Becton Dickinson) using 488nm (calcein AM, 530/30 filter), 640nm (TO-PRO-3, 670/30 filter) and 405 nm (Vioblue, 450/50 filter) lasers. Non-stained controls were included with all tumours. CD45-positive cells were counterselected for the St. Jude samples only and viable medulloblastoma cells were identified by staining positive with calcein AM but negative for TO-PRO-3. Forward scatter area (FSC-A) versus side scatter width (SSC-W) criteria were used to discriminate doublets and select single cells. Single cells were sorted into 96-well plates containing cold TCL buffer (Qiagen, 1031576) containing 1% beta-mercaptoethanol, snap frozen on dry ice, and then stored at −80°C prior to whole transcriptome amplification, library preparation and sequencing.
6
Synthesis of Gold Nanoparticles
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Gold(III) chloride trihydrate (HAuCl4·3H2O, ≥49% Au basis), cetyltrimethylammonium bromide (CTAB, ≥99%), sodium borohydride (NaBH4, ≥98%), silver nitrate (AgNO3, ≥99.0%), ascorbic acid (AA, ≥99.0%) and sodium hydroxide (NaOH, ≥98%) were purchased from Sigma Aldrich. Tetraethyl orthosilicate (TEOS, ≥99.0%) and Methanol (≥99%) was products of Macklin. Hydrochloric acid (HCl, 35–37%) were purchased from Acros Organics. Ethanol (≥99.7%) was purchased from Ghtech. Phosphate-Buffered Saline (PBS) were procured from Gibco, Switzerland. Propidium Iodide (PI) and Calcein-AM were purchased from Becton Dickinson Pharmingen, USA. Deionized water used throughout all experiments was ultra-pure Milli-Q water with an electrical resistivity of 18.25 MΩ·cm (25 °C). Carbon support films (300 eyes) for TEM purchased from Beijing XXBR Technology Co., Ltd.
7
Fluorescence-Activated Sorting of Viable Tumor Cells
8
Isolation and Labeling of Human Neutrophils
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Heparinized human blood was mixed with 1.5% dextran/0.9% NaCl and allowed to settle undisturbed for 30 min. The leukocyte-rich plasma was layered over a histopaque 1077 density gradient (Sigma) and centrifuged at 1300 × g for 30 min. The cell pellet containing PMNs and erythrocytes was treated with hypotonic lysis buffer and washed with HBSS buffer without Ca2+/Mg2+. The preparation contained ~95% neutrophils, as judged by morphological examination and cells were >90% viable, as determined by Trypan blue dye exclusion. PMNs were washed 3× and re-suspended in HBSS buffer without Ca2+/Mg2+ at 1 × 107 cells/ml (Gibco). Prior to their use, calcein AM (1:1000, Becton Dickinson) was added to the suspension of PMN as described (Ruiz-Perez et al., 2011 (link)). The cells were incubated for 30 min at 37°C, and washed up to 3× with PBS before being re-suspended in 1 ml HBSS buffer without Ca2+/Mg2+.
9
Single-cell Immune Cell Sorting and Sequencing
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Following the single-cell suspension techniques of flow cytometry, tumor cells were blocked in 1% bovine serum albumin in phosphate-buffered saline solution (1% BSA/PBS). Cell suspensions were subsequently stained for flow cytometry for 30 min at 4 °C using antibodies specific for CD45 [30F11]-VioBlue from Miltenyi, CD3 [145-2C11]-PE from Biolegends, and CD31 [MEC 13.3]-PE from BD Biosciences. Cells were washed with cold PBS and then incubated for 15 min in 1.5 mL of 1% BSA/PBS containing 1 μM calcein AM (Life Technologies) and 0.33 μM TO-PRO-3 iodide (Life Technologies). Sorting was performed with the FACS Aria Fusion Special Order System (Becton Dickinson) using 488-nm (calcein AM, 530/30 filter; CD3-PE, 585/42 filter), 640-nm (TO-PRO-3, 670/14 filter), and 405-nm (CD45-VioBlue, 450/50 filter) lasers. Standard, strict forward scatter height vs. area criteria were used to discriminate doublets and gate-only singleton cells. Viable cells were identified by staining positive with calcein AM but negative for TO-PRO-3. We sorted individual, viable, CD45+CD3− and CD45+CD3+ immune, and CD45− nonimmune single cells into 96-well plates containing cold TCL buffer (QIAGEN) with 1% beta-mercaptoethanol. Plates were frozen on dry ice immediately after sorting and stored at −80 °C prior to whole transcriptome amplification, library preparation, and sequencing.
10
Resveratrol Inhibits Cell Migration
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The cells (in log phase growth stage) were harvested, centrifuged, suspended in FBS-free culture medium to the final density of 8 × 105 cell/ml. 250 pl of the prepared cells suspension (2 × 105 cell) was added on the 3 µm Fluoro Blok inserts covered with Matrigel TM (Becton Dickinson) and placed in a 24-well plate. Chambers under the inserts were filled with 750 µl of culture medium (DMEM, 4.5 g/ml glucose with L-glutamine, 5% FBS, 50 U/ml penicillin, 50 µg/ml streptomycin). 10 or 50 µM of resveratrol were added to the lower chamber. As a control, culture medium without resveratrol was used. As a negative control, FBS-free culture medium was used. After 24 h incubation the medium was discarded and 4 µM of fluorescent dye Calcein AM (Becton Dickinson) in PBS was added. One hour later both plate and inserts were washed twice with PBS and fluorescence (excitation 485 nm and emission 510 nm) was measured directly in the inserts using the FLUOstar Omega reader with “bottom optic ” option. The results were shown as the relative fluorescence units - RFU (mean ± SEM).
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