Enriched reticulocytes and normocytes were resuspended to 1% hematocrit with PBS and stained with CellTracker Deep Red (at 2 µM; Invitrogen #C34565) and CellTrace Oregon Green (at 15 µM; Invitrogen #C34555), respectively. After 30 mins of staining at 37°C, warm FBS was added to stop the staining reaction. Cells were then washed twice with media, with another 10 mins incubation during the second wash. Stained reticulocytes and normocytes were mixed to the preferred ratios (10:90, 30:70, 50:50, 70:30, or 90:10) and resuspended in parasite media to 2% hematocrit. Enriched late-stage parasites were then added at around 5-10% final parasitemia and the cells were incubated at 37°C for 12 hours. At Time = 0 hr (i.e. after the addition of parasites), aliquots of samples were mixed with PBS and stained with 8 µM Hoechst for 15-20 mins. 300 µL PBS was then added to quench the reaction and the samples were immediately acquired. The staining and flow cytometry acquisition was repeated at Time = 12 hr. This 12-hour incubation allows sufficient time for the trophozoites and schizonts to develop and release merozoites, and yet not too long until a second round of invasion occurs [e.g. the asexual cycle of different P. yoelii strains vary between 18-24 hours (Killick-Kendrick and Warben, 1968 (link); Gautret et al., 1994 (link))] or for the target reticulocytes to mature into normocytes.
Celltrace oregon green
Manufactured by Thermo Fisher Scientific
Sourced in New Zealand
CellTrace Oregon Green is a fluorescent dye used for cell labeling and tracking applications. It provides bright, photostable green fluorescence that can be used to monitor cell division, migration, and other cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
C12fdg, by Thermo Fisher Scientific (3 mentions)
Celltrace far red, by Thermo Fisher Scientific (3 mentions)
Chicken anti rabbit alexa 594, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (3 mentions)
Anti hmga2 and p21, by Cell Signaling Technology (3 mentions)
Alexa 488 chicken anti mouse, by Thermo Fisher Scientific (3 mentions)
α tubulin, by Abcam (3 mentions)
Anti h3k9me3, by Merck Group (3 mentions)
Vectashield, by Vector Laboratories (3 mentions)
Ba lomycin a1, by Thermo Fisher Scientific (2 mentions)
Axio plan 2 uorescence microscope, by Zeiss (2 mentions)
Celltracker deep red, by Thermo Fisher Scientific (1 mentions)
Axioplan 2, by Zeiss (1 mentions)
Bafilomycin a1, by Thermo Fisher Scientific (1 mentions)
5 protocols using celltrace oregon green
1
Reticulocyte-Normocyte Co-culture Assay
2
Cell Trace Oregon Green Labeling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For division tracking, Teffs were labeled with a final concentration of 20 μM Cell Trace Oregon Green (Invitrogen) by incubation for 10 min at 37°C at a cell density of 107 cells/mL in phosphate-buffered saline (PBS) with 10% bovine-serum albumin (BSA).
3
Senescence Detection by Flow Cytometry and Immunostaining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Senescence was determined either by ow cytometry or immunostaining as two independent methods.
For the ow cytometric analysis, cells were incubated for 30 minutes in Ba lomycin A1 and afterwards C 12 FDG (Invitrogen, Auckland, New Zealand) was added for 60 min at 37°C. For senescence analysis by immunostaining anti-H3K9me3 (Millipore, Darmstadt, Germany), anti-HMGA2 and p21 (Cell Signaling, Leiden, Netherlands) antibodies were used. Cytoskeleton was stained by α-tubulin (Abcam, Cambridge, UK). Secondary antibodies were Alexa 488 chicken anti mouse or Alexa 594 chicken anti rabbit (Molecular Probes, Darmstadt, Germany). Phagocytosis experiments were performed using CellTrace Oregon Green (CTOG) (Invitrogen, Auckland New Zealand) for the living cells and CellTrace Far Red (CTFR) (Invitrogen, Auckland, New Zealand) for heat (56°C) for camptothecin treated cells. Cell nuclei were stained by DAPI (Roche, Grenzach-Wyhlen, Germany) and slides were mounted using Vectashield (Vector Laboratories Inc, Burlingame, USA). Cell images were acquired by a Zeiss Axio Plan 2 uorescence microscope (Zeiss, Göttingen, Germany). Nuclear size was analyzed with Biomas software (MSAB, Erlangen, Germany).
For the ow cytometric analysis, cells were incubated for 30 minutes in Ba lomycin A1 and afterwards C 12 FDG (Invitrogen, Auckland, New Zealand) was added for 60 min at 37°C. For senescence analysis by immunostaining anti-H3K9me3 (Millipore, Darmstadt, Germany), anti-HMGA2 and p21 (Cell Signaling, Leiden, Netherlands) antibodies were used. Cytoskeleton was stained by α-tubulin (Abcam, Cambridge, UK). Secondary antibodies were Alexa 488 chicken anti mouse or Alexa 594 chicken anti rabbit (Molecular Probes, Darmstadt, Germany). Phagocytosis experiments were performed using CellTrace Oregon Green (CTOG) (Invitrogen, Auckland New Zealand) for the living cells and CellTrace Far Red (CTFR) (Invitrogen, Auckland, New Zealand) for heat (56°C) for camptothecin treated cells. Cell nuclei were stained by DAPI (Roche, Grenzach-Wyhlen, Germany) and slides were mounted using Vectashield (Vector Laboratories Inc, Burlingame, USA). Cell images were acquired by a Zeiss Axio Plan 2 uorescence microscope (Zeiss, Göttingen, Germany). Nuclear size was analyzed with Biomas software (MSAB, Erlangen, Germany).
4
Senescence and Phagocytosis Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Senescence was determined either by flow cytometry or immunostaining as two independent methods. For the flow cytometric analysis, cells were incubated for 30 minutes in Bafilomycin A1 and afterwards C 12 FDG (Invitrogen, Auckland, New Zealand) was added for 60 min at 37°C. For senescence analysis by immunostaining anti-H3K9me3 (Millipore, Darmstadt, Germany), anti-HMGA2 and p21 (Cell Signaling, Leiden, Netherlands) antibodies were used. Cytoskeleton was stained by α-tubulin (Abcam, Cambridge, UK). Secondary antibodies were Alexa 488 chicken anti mouse or Alexa 594 chicken anti rabbit (Molecular Probes, Darmstadt, Germany).
Phagocytosis experiments were performed using CellTrace Oregon Green (CTOG) (Invitrogen, Auckland New Zealand) for the living cells and CellTrace Far Red (CTFR) (Invitrogen, Auckland, New Zealand) for heat (56°C) for camptothecin treated cells. Cell nuclei were stained by DAPI (Roche, Grenzach-Wyhlen, Germany) and slides were mounted using Vectashield (Vector Laboratories Inc, Burlingame, USA). Cell images were acquired by a Zeiss Axio Plan 2 fluorescence microscope (Zeiss, Göttingen, Germany). Nuclear size was analyzed with Biomas software (MSAB, Erlangen, Germany).
Phagocytosis experiments were performed using CellTrace Oregon Green (CTOG) (Invitrogen, Auckland New Zealand) for the living cells and CellTrace Far Red (CTFR) (Invitrogen, Auckland, New Zealand) for heat (56°C) for camptothecin treated cells. Cell nuclei were stained by DAPI (Roche, Grenzach-Wyhlen, Germany) and slides were mounted using Vectashield (Vector Laboratories Inc, Burlingame, USA). Cell images were acquired by a Zeiss Axio Plan 2 fluorescence microscope (Zeiss, Göttingen, Germany). Nuclear size was analyzed with Biomas software (MSAB, Erlangen, Germany).
5
Senescence Analysis by Flow Cytometry and Immunostaining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Senescence was determined either by ow cytometry or immunostaining as two independent methods. For the ow cytometric analysis, cells were incubated for 30 minutes in Ba lomycin A1 and afterwards C 12 FDG (Invitrogen, Auckland, New Zealand) was added for 60 min at 37°C. For senescence analysis by immunostaining anti-H3K9me3 (Millipore, Darmstadt, Germany), anti-HMGA2 and p21 (Cell Signaling, Leiden, Netherlands) antibodies were used. Cytoskeleton was stained by α-tubulin (Abcam, Cambridge, UK). Secondary antibodies were Alexa 488 chicken anti mouse or Alexa 594 chicken anti rabbit (Molecular Probes, Darmstadt, Germany). Phagocytosis experiments were performed using CellTrace Oregon Green (CTOG) (Invitrogen, Auckland New Zealand) for the living cells and CellTrace Far Red (CTFR) (Invitrogen, Auckland, New Zealand) for heat (56°C) for camptothecin treated cells. Cell nuclei were stained by DAPI (Roche, Grenzach-Wyhlen, Germany) and slides were mounted using Vectashield (Vector Laboratories Inc, Burlingame, USA). Cell images were acquired by a Zeiss Axio Plan 2 uorescence microscope (Zeiss, Göttingen, Germany). Nuclear size was analyzed with Biomas software (MSAB, Erlangen, Germany).
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!