The isolated cells were identified by flow cytometry assay with lineage-specific markers, including anti-CD90 (ab33694, Abcam, UK) and anti-CD31 (ab33858; Abcam, UK). Isotype controls (ab91357, ab91356; Abcam, UK) were used for anti-CD90 and anti-CD31, respectively. TDSCs (5 × 105) at P3 were incubated with fluorescein-conjugated anti-rat monoclonal antibodies for 1 h at 4°C and centrifuged at 1000 rpm for 5 min. The stained cells were resuspended in 500 μL ice-cold PBS containing 10% FBS and analyzed by FACs (FACSCalibur, Becton Dickinson) [23 (link)].
Ab91357
Manufactured by Abcam
Sourced in United Kingdom
Ab91357 is a lab equipment product. It is a recombinant antibody that recognizes a specific target. The core function of this product is to detect and bind to the target.
Automatically generated - may contain errors
Lab products found in correlation
Cytoflex, by Beckman Coulter (2 mentions)
Cytexpert 2, by Beckman Coulter (2 mentions)
Ab112068, by Abcam (2 mentions)
Facscalibur, by BD (1 mentions)
Ab33858, by Abcam (1 mentions)
Ab91356, by Abcam (1 mentions)
Ab33694, by Abcam (1 mentions)
Positive control, by Merck Group (1 mentions)
Annexin 5, by Thermo Fisher Scientific (1 mentions)
Cytoflex s, by Beckman Coulter (1 mentions)
5 protocols using ab91357
1
Flow Cytometry Characterization of TDSCs
2
Tumor Marker Analysis and Cell Death Assay
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To examine the percentage of
selected tumor markers, single cells were resuspended in a blocking
solution (ice-cold PBS containing 10% FBS and 1% sodium azide) at
a concentration of 1 × 106 cells/mL. They were then
incubated at 4 °C for 30 min in the dark with 0.1 μg/mL
α-EpCAM-PE CD36 antibody (ab112068, Abcam; λex = 488 nm, λem = 575) and isotype-matched control
PE mouse IgG1 [B11/6] (ab91357, Abcam; λex = 488
nm, λem = 575). Cells were then washed three times
by centrifugation (300g, 5 min) and resuspended in
0.5 mL of blocking solution.
Cell death induced by the different
treatments was determined using an Apoptosis Kit (Thermo Fisher) based
on annexin V-FITC and propidium iodide (PI). Cells were resuspended
in the annexin V-FITC-binding buffer supplied with the kit containing
1 μg/mL of PI and annexin V-FITC 7 mM for 15 min (0.1 mL total
volume). At the end of the incubation, 0.4 mL of the binding buffer
was added to each sample, and the fluorescence of the cells was measured
(annexin V-FITC: λex = 488 nm, λem = 525 ± 40 nm; PI: λex = 488 nm, λem = 610 ± 20 nm).
The nanovector accumulation in
the tumor cells was analyzed using
the PCy7 channel (λex = 488 nm, λem = 780 ± 60 nm). All flow cytometry analyses were performed
using a Cytoflex (Beckmann Coulter), and data were interpreted by
using the CytExpert 2.5 software (Beckman Coulter).
selected tumor markers, single cells were resuspended in a blocking
solution (ice-cold PBS containing 10% FBS and 1% sodium azide) at
a concentration of 1 × 106 cells/mL. They were then
incubated at 4 °C for 30 min in the dark with 0.1 μg/mL
α-EpCAM-PE CD36 antibody (ab112068, Abcam; λex = 488 nm, λem = 575) and isotype-matched control
PE mouse IgG1 [B11/6] (ab91357, Abcam; λex = 488
nm, λem = 575). Cells were then washed three times
by centrifugation (300g, 5 min) and resuspended in
0.5 mL of blocking solution.
Cell death induced by the different
treatments was determined using an Apoptosis Kit (Thermo Fisher) based
on annexin V-FITC and propidium iodide (PI). Cells were resuspended
in the annexin V-FITC-binding buffer supplied with the kit containing
1 μg/mL of PI and annexin V-FITC 7 mM for 15 min (0.1 mL total
volume). At the end of the incubation, 0.4 mL of the binding buffer
was added to each sample, and the fluorescence of the cells was measured
(annexin V-FITC: λex = 488 nm, λem = 525 ± 40 nm; PI: λex = 488 nm, λem = 610 ± 20 nm).
The nanovector accumulation in
the tumor cells was analyzed using
the PCy7 channel (λex = 488 nm, λem = 780 ± 60 nm). All flow cytometry analyses were performed
using a Cytoflex (Beckmann Coulter), and data were interpreted by
using the CytExpert 2.5 software (Beckman Coulter).
3
Calreticulin Upregulation Kinetics
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5 × 105 cells were seeded in a T25 cm2 flask with 5 mL DMEM. To determine the kinetics of calreticulin upregulation, ID8 cells were treated with 1 Gy radiation or doxorubicin (positive control, 25 μmol/L; Sigma) and harvested 4, 6, 12, and 24 hours after exposure as previously described, washed twice with cold PBS, followed by staining with a calreticulin-specific antibody (Abcam; catalog no. ab83220, RRID:AB_1859755), Annexin V (Thermo Fisher Scientific; catalog no. 88-8007-72, RRID:AB_2575165), which recognizes phosphatidylserine on the surface of apoptotic cells, plus vital dye 4,6-diamidino-2-phenylindole (DAPI), which stains dead cells. Isotype-matched IgG antibody was used as a negative control (Abcam; catalog no. ab91357, RRID:AB_2888649), and the analysis was limited to living (DAPI-negative) tumor cells (104 (link)).
4
Quantifying Tumor Marker Expression and Cell Death
5
Nanoscale Flow Cytometry Analysis of EVs
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Nanoscale flow cytometry analysis of EVs was performed as we have previously described.6,7 (link) Briefly, 2.5–10 μL of filtered plasma or isolated SEC EV-fraction were incubated with 37.5–150 ng of CF405M fluorescently labeled CD9 antibody (Abcam, ab123624), or 37.5–150 ng of CF405M mouse IgG2a antibody isotype control (Abcam, ab126036), for 30 minutes at room temperature in the dark, followed by the addition of 75–300 μL of 0.02 μm filtered PBS and transferred into a 96-well plate. Dual labeling was performed by the addition of 1 μg of PE labeled CD41 (Abcam, ab134372) or 1 μg PE mouse IgG1 isotype control (Abcam, ab91357). Samples were analyzed using the CytoFLEX S (Beckman Coulter) instrument for 60 seconds on slow (10 μL min−1) using 405 nm violet side scatter (VSSC) trigger. To prevent cross-contamination between samples, a PBS wash was done between each sample. The VSSC trigger threshold was set at 1027. Gain settings used: FITC – 500, BV421 – 77, and PE – 135. Isotype controls were used to guide manual gating of populations of interest.
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