iRTN-1c MCA205 cells were treated (or not) by 1 µM MTX, 150 µM CDDP, 0.3 µM TET, or CDDP+TET, for 24 h, prior to cell harvest and subsequent wash with ice cold PBS. After 30 min incubation with an anti-CRT antibody (Abcam) in cold blocking buffer (5% FBS, v/v in PBS) on ice, cells were further washed and incubated with AlexaFluor®488-conjugated secondary antibody (Life Technologies) in blocking buffer (for 30 min). Cells were finally washed and maintained in cold PBS with 1 μg/mL PI and samples were analyzed by means of a FACS Calibur cytofluorometer (BD Biosciences). Isotype-matched IgG antibodies (Cell Signaling Technology) were used a negative staining control, and the analysis was performed exclusively on non-permeabilized (PI-) cells. Data were statistically evaluated by means of the Cell Quest Software package (BD Biosciences).
Anti crt antibody
Manufactured by Abcam
Sourced in United States
The Anti-CRT antibody is a tool used in research applications to detect and study the Calreticulin (CRT) protein, which is involved in various cellular processes. This antibody can be used in techniques such as Western blotting, immunohistochemistry, and immunocytochemistry to identify and analyze the expression of CRT in different samples.
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Lab products found in correlation
Cellquest software package, by BD (1 mentions)
Isotype matched igg antibodies, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Anti mouse igg fitc, by Merck Group (1 mentions)
Anti rabbit igg fitc, by Merck Group (1 mentions)
Anti hmgb1 primary antibody, by Abcam (1 mentions)
Facscanto 2, by BD (1 mentions)
Fitc conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Anti herg, by Alomone (1 mentions)
Propranolol prop, by Merck Group (1 mentions)
Anti cd8 pe, by BioLegend (1 mentions)
Anti cd80 pe, by BioLegend (1 mentions)
Hemin, by Merck Group (1 mentions)
Anti cd3 fitc, by BioLegend (1 mentions)
Anti cd86 apc, by BioLegend (1 mentions)
Anti foxp3 pe, by BioLegend (1 mentions)
Anti cd11c fitc, by BioLegend (1 mentions)
Anti pd 1, by BioXCell (1 mentions)
7 protocols using anti crt antibody
1
Quantification of Cell Surface Calreticulin
2
Detecting Immunogenic Cell Death by CRT
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The presence of CRT in the plasma membrane characterizes substances that induce the immunogenic type of cell death (Kepp et al. 2011 (link)). CRT externalization could work as a signal for immunological cells (Zitvogel et al. 2010 (link)). The presence of CRT in the plasma membrane has been determined by an antiCRT antibody conjugated with the fluorochrome phycoerythrin (Abcam, Great Britain). 1 × 106 cells were incubated with antibody diluted 1:100 for 30 min at 4 °C and analyzed in flow cytometer.
3
Quantifying HMGB1 and CRT in S180 Cells
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After PTT treatment, S180 cells were washed twice with 1× PBS and fixed with 4% paraformaldehyde (PFA) for 5 min. Cells were centrifuged at 1500 rpm for 3 min and washed again with PBS.
To measure intracellular HMGB1, S180 cells were incubated for 15 min with permeabilization buffer and washed with PBS (containing 0.1% BSA and 0.1% Triton-X). Then, cells were incubated in the dark at room temperature for 1 h with anti-HMGB1 primary antibody (Abcam, Cambridge, MA, USA) at 1:103. S180 cells were then washed and stained with anti-mouse IgG-FITC (Sigma-Aldrich) for 30 min. Finally, cells were washed twice with PBS, centrifuged for 5 min at 1500 rpm, resuspended in PBS, and analyzed by flow cytometry (FACSCanto II, BD Biosciences).
To measure CRT externalization, S180 cells containing RBCm-IR-Mn were incubated for 1 h with anti-CRT antibody (Abcam Cambridge, MA) at 1:500 in 0.1% BSA/PBS. Next, cells were washed and stained with a secondary antibody (anti-rabbit IgG-FITC, Sigma-Aldrich) for 30 min. Samples were washed, centrifuged, resuspended in PBS, and analyzed by flow cytometry (FACSCanto II, BD Biosciences).
To measure intracellular HMGB1, S180 cells were incubated for 15 min with permeabilization buffer and washed with PBS (containing 0.1% BSA and 0.1% Triton-X). Then, cells were incubated in the dark at room temperature for 1 h with anti-HMGB1 primary antibody (Abcam, Cambridge, MA, USA) at 1:103. S180 cells were then washed and stained with anti-mouse IgG-FITC (Sigma-Aldrich) for 30 min. Finally, cells were washed twice with PBS, centrifuged for 5 min at 1500 rpm, resuspended in PBS, and analyzed by flow cytometry (FACSCanto II, BD Biosciences).
To measure CRT externalization, S180 cells containing RBCm-IR-Mn were incubated for 1 h with anti-CRT antibody (Abcam Cambridge, MA) at 1:500 in 0.1% BSA/PBS. Next, cells were washed and stained with a secondary antibody (anti-rabbit IgG-FITC, Sigma-Aldrich) for 30 min. Samples were washed, centrifuged, resuspended in PBS, and analyzed by flow cytometry (FACSCanto II, BD Biosciences).
4
Flow Cytometric Analysis of Cell Surface Calreticulin
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Cells were trypsinized and washed twice with FACS buffer, and then incubated with anti-CRT antibody (Abcam, Cambridge, MA, USA) at 1:200 dilution in 100 μL FACS buffer at 4 °C for 1 h. Then, cells were washed and stained with FITC-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) for 30 min, followed by staining with propidium iodide (PI, Invitrogen) and acquisition by flow cytometry, and the fluorescent intensity of stained live cells was gated on propidium iodide (PI)-negative cells.
5
Antibody and Inhibitor Sources for ER Stress
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Rabbit polyclonal anti-hERG was purchased from Alomone (Alomone, Israel). Anti-CNX antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-CRT antibody was purchased from Abcam (Abcam, U.S.A.). Rabbit polyclonal anti-ATF6 was purchased from Active Motif (Active Motif, U.S.A.). The proteasome inhibitor of N-acetyl-l -leucyl-l -leucyl-l -norleucinal (ALLN) was purchased from Calbiochem, Propranolol (Prop) was purchased from Sigma (Sigma, U.S.A.).
6
Fabrication of PLGA-based Nanoparticles
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LOX, hemin, poly (d ,l -lactic-co-glycolic acid) (PLGA), and polyvinyl alcohol (PVA) were obtained from Sigma-Aldrich. Dichloromethane (DCM), sodium bicarbonate (NaHCO3), and calcium chloride (CaCl2) were obtained from Sinopharm Chemical Reagent Co. Anti-HMGB1 antibody (catalog: 70-ab40050-100) was obtained from MultiSciences. Anti-CRT antibody (catalog: ab2907) was obtained from Abcam. Alexa 488-conjugated secondary antibody (catalog: 111-545-003) was obtained from Jackson. Antibodies for flow cytometry assays including anti-CD3-FITC (Biolegend, clone 17A2, catalog: 100204), anti-CD4-APC (Biolegend, clone GK1.5, catalog: 100412), anti-CD8-PE (Biolegend, clone 53-6.7, catalog: 100708), and anti-Foxp3-PE (Biolegend, clone MF-14, catalog: 126404), anti-CD11c-FITC (Biolegend, clone N418, catalog: 117306), anti-CD80-PE (Biolegend, clone 16-10A1, catalog: 104708), and anti-CD86-APC (Biolegend, clone GL-1, catalog: 105012) were obtained from Biolegend or eBioscience as indicated and diluted at 1:300 for cell staining. Anti-PD-1 (catalog: BE0146) was purchased from BioXcell.
7
Calreticulin Expression in Preeclampsia
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Paraffin-embedded blocks of placental tissue samples (patients with preeclampsia: n = 18; gestational age-matched controls: n = 22) were cut into 3-μm-thick sections, deparaffinized, and rehydrated. We then blocked endogenous peroxidase activity with 0.3% hydrogen peroxide in methanol. For antigen retrieval, we boiled the slides in 1 mM ethylenediaminetetraacetic acid (pH 8.0) in a pressure cooker for 10 min. We incubated the sections with an anti-CRT antibody (Abcam) at a dilution of 1:5000. We detected signals by using the simple stain MAX-PO reagent (Nichirei Biosciences, Tokyo, Japan) and 3,3-diaminobenzidine as the substrate. We used Mayer’s hematoxylin solution for counterstaining and scored the staining intensity as 0 = none, 1 = weak, 2 = moderate, and 3 = strong staining. We graded the percentage of positive cells as 0: no positive cells, 1: <30%, 2: 30–80%, or 3: >80% and then used the following equation to calculate the immunoreactive score (IRS): IRS = staining intensity × percentage of positive cells, according to previous reports [16 (link),27 (link)].
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