Here, intra-peritoneal injection of leukemia cells is utilized to establish and propagate acute myeloid leukemia (AML) in mice. This new method is effective in the serial transplantation of AML cells and can serve as an alternative for those who may experience difficulties and inconsistencies with intravenous injection in mice.
There is an unmet need for novel therapies to treat acute myeloid leukemia (AML) and the associated relapse that involves persistent leukemia stem cells (LSCs). An experimental AML rodent model to test therapies based on successfully transplanting these cells via retro-orbital injections in recipient mice is fraught with challenges. The aim of this study was to develop an easy, reliable, and consistent method to generate a robust murine model of AML using an intra-peritoneal route. In the present protocol, bone marrow cells were transduced with a retrovirus expressing human MLL-AF9 fusion oncoprotein. The efficiency of lineage negative (Lin–) and Lin–Sca-1+c-Kit+ (LSK) populations as donor LSCs in the development of primary AML was tested, and intra-peritoneal injection was adopted as a new method to generate AML. Comparison between intra-peritoneal and retro-orbital injections was done in serial transplantations to compare and contrast the two methods. Both Lin– and LSK cells transduced with human MLL-AF9 virus engrafted well in the bone marrow and spleen of recipients, leading to a full-blown AML. The intra-peritoneal injection of donor cells established AML in recipients upon serial transplantation, and the infiltration of AML cells was detected in the blood, bone marrow, spleen, and liver of recipients by flow cytometry, qPCR, and histological analyses. Thus, intra-peritoneal injection is an efficient method of AML induction using serial transplantation of donor leukemic cells.
Acute myeloid leukemia (AML) is a type of hematologic malignancy of diverse etiology with poor prognosis1. The generation of AML animal models lays the foundation for the understanding of its complex variations and pathobiology in an effort to discover novel therapies2. Leukemogenesis in mice involves the transplantation of donor cells expressing fusion oncoproteins, including fusions involving the mixed lineage leukemia (MLL) gene to potently induce AML, to mimic the disease in humans3. Various cellular origins of donor cells have been reported in the transplantation of MLL gene-associated AML4, with very little being known about the cells responsible for the disease origin.
Multiple routes have been developed for transplantation in mice; rather than an intra-femoral injection, which directly introduces mutant donor cells into bone marrow5, an intravenous injection that utilizes the venous sinus plexus, tail vein, and jugular vein has been widely used to generate murine AML models6,7,8,9. In the case of retro-orbital (r.o.) injection, various inherent disadvantages, such as volume limitation, high technical demand, few chances for repeated attempts or error, and potential ocular injuries, have been major stumbling blocks with limited or no viable alternatives7. Tail vein injection can have similar problems besides local injuries; to facilitate the procedure, mice often need to be warmed up to dilate their tail veins10. It is also hard to locate the tail vein without an additional light source, particularly in the C57BL/6 strain of mice. For jugular vein injection, research personnel require sufficient training to locate the vein and limit possible complications. In addition, both venous sinus and jugular vein injections need to be performed under anesthesia, which adds another level of complexity. Thus, it is tempting to explore new routes for transplantation to facilitate the establishment of AML murine models.
Intra-peritoneal (i.p.) injection is commonly used to administer drugs, dyes, and anesthetics11,12,13,14,15; it has also been used to introduce hematopoietic cells for ectopic hematopoiesis16 and to transplant bone marrow-derived mesenchymal stem cells in various mouse models17,18,19,20,21. However, it has been infrequently used to establish hematopoietic malignancies in mice, particularly to study AML disease progression.
The present study describes the feasibility of i.p. injection in the generation of AML mouse models, in addition to comparing the transplantation efficiency of lineage negative (Lin–) and Lin–Sca-1+c-Kit+ (LSK) populations as donor cells. These findings provide a simple and efficient way to generate experimental models of AML and related myeloid leukemias. Such a method has the potential to further our understanding of the disease mechanisms as well as provide a relatively easy model to test experimental therapies.
All experiments were preapproved by the Institutional Animal Care and Use Committee at the Pennsylvania State University.
1. Preparation of buffers and reagents
2. Plasmid transformation
3. Transfection of Phoenix Ecotropic (pECO) cells
4. Lentiviral transduction
5. Serial transplantation (Figure 1)
NOTE: Primary recipient mice were 8-10-week-old male C57BL6/J mice (CD45.2). They were provided water ad libitum containing antibiotics to prevent opportunistic digestive infections, from 3 days prior to transplantation to 7 days post transplantation. Primary recipient mice were sub-lethally irradiated (4.75 Gy) 3 h before transplantation25. Isoflurane was not applied to mice with intra-peritoneal injection.
Figure 1: Schematic of MLL-AF9 viral transduction in bone marrow HSCs and serial transplantation (1˚, 2˚, and 3˚). Sorting of Sca-1 and c-Kit double positive population by using a cell sorter shown in the dotted shade box is considered optional, should resources allow. The figure was created using BioRender (https://biorender.com/). Please click here to view a larger version of this figure.
6. Intra-peritoneal lavage
7. Histological analysis 28
8. Performing semi-quantitative PCR (qPCR)
9. Data processing
Comparison of the transplantation efficiency of murine AML cells using r.o. and i.p. routes of transplantation
Previously, the establishment of 1˚ AML was reported in recipient mice retro-orbitally transplanted with MLL-AF9-transduced LSK cells, and the transplantability of 1˚ AML cells was demonstrated by serial transplantation30. The present study is the first to evaluate the possibility of using bone marrow Lin– cells to perform transplantation. The presence of aberrant leukocytosis (Figure 2A) and increased infiltration of leukemic cells (CD45.1+) in the bone marrow and spleen (Figure 2B) support the feasibility of using bone marrow Lin– cells to generate 1˚ AML. To compare the disease induction period, two donor mice per recipient were euthanized to isolate either LSK or Lin– cells. From the results, no significant differences were observed in 1˚ recipients transplanted with LSK or Lin– cells (Figure 2C).
During the examination of metastasis of 1˚ AML, it was discovered that the AML cells spread in the abdominal cavity of 1˚ recipients by MLL-AF9-transduced Lin– cells (Supplementary Figure 1). This finding inspired the exploration to test whether i.p. injection could be adopted in the generation of murine AML, which might serve as a new and easier route of transplantation. Due to the success of using the bone marrow Lin– population as AML donor cells, current data confirmed the establishment of 1˚ AML via i.p. injection of MLL-AF9-transduced bone marrow Lin– cells, as seen in the form of leukocytosis (Figure 2D) and the presence of AML cells (CD45.1+) in the bone marrow and spleen of recipient mice (Figure 2E). However, transplantation via i.p. injection took longer to develop AML than via r.o. injection, despite the recipients receiving an equal number of donor cells (Figure 2F). Besides, mice transplanted retro-orbitally with more Lin– cells (5.125 x 106 cells/mouse) in Figure 2F seemed to develop AML faster than those transplanted with fewer Lin– cells (3.69 x 106 cells/mouse) in Figure 2C.
Owing to the inherent inconsistency of incubation time in 1˚ AML recipients (Figure 2F), an attempt was made to test the i.p. transplantation in 2˚ recipients. For 2˚ transplantation, i.p. injection was performed with 8 x 105 AML cells isolated from intra-peritoneally transplanted 1˚ recipients. The 2˚ recipients showed leukocytosis (Figure 2G) and significant hepatosplenomegaly (Figure 2H) in less than 1 month post transplantation, a clear advancement from 1˚ transplantation. Consistent with this observation, AML cells (RFP–) were also detected in the peripheral blood, bone marrow, and spleen (Figure 2I), as well as in the peritoneal cavity (Supplementary Figure 1). Besides, the expression of oncogene KMT2A in the blood, spleen, and liver of 2˚ recipients also confirmed the successful generation of AML (Figure 2J). Moreover, histological analysis further demonstrated the infiltration of leukemic cells in the spleen and liver of intra-peritoneally transplanted mice (Figure 2K). These data confirm that i.p. injection-generated 1˚ AML cells are transplantable in 2˚ recipients. Furthermore, i.p. injection of 8 x 105 AML cells per mouse into recipients achieved comparable engraftment to r.o. injection of 8 x 105 AML cells per mouse into recipients (Figure 2L).
One of the key features of LSCs is serial transplantability; thus, to further ascertain the establishment of AML in 2˚ recipients, this protocol attempted to verify if AML cells from 2˚ i.p. transplantation remained serially transplantable, as well as the feasibility of stem cell transplantation by i.p. injection in 3˚ transplantation. AML cells isolated from either bone marrow (8 x 105 leukemic cells/mouse in 0.5 mL of PBS) or i.p. lavage (4 x 105 leukemic cells/mouse in 0.5 mL of PBS) of 2˚ recipients were injected into 3˚ recipients. Although being transplanted with donor cells generated from different sources, 3˚ recipient mice acutely exhibited AML signs (within 3 and 4 weeks for bone marrow cells and i.p. cells, respectively), including leukocytosis (Figure 3A) and hepatosplenomegaly (Figure 3B), the presence of leukemic cells in the blood, bone marrow, and spleen (Figure 3C), and the expression of KMT2A (Figure 3D). Histological observation of the femur and spleen further demonstrated the infiltration of leukemic cells (Figure 3E). As a comparison, r.o. injection of leukemic splenocytes (4 x 105 cells) from 2˚ recipients was also equally capable of engrafting and developing AML in 3˚ recipients, characterized by leukocytosis (Supplementary Figure 2A), hepatosplenomegaly (Supplementary Figure 2B), and the infiltration of AML cells in the blood, bone marrow, and spleen (Supplementary Figure 2C).
Intra-peritoneal transplantation is effective even for 3˚ transplantation of AML cells
Compared to the 2˚ and 3˚ transplantations, a conclusion could easily be made that 3˚ transplantation (Figure 3F) progressed much faster than 2˚ transplantation (Figure 2L) for both i.p. and r.o. injections. Notably, i.p. injection of 4 x 105 AML cells/mouse developed AML later than r.o. injection of same number of AML cells for 6 days (Figure 3F), possibly indicating the time spent on the migration from the peritoneal cavity to the bone marrow niche. Interestingly, the high frequency of leukemic cells in the peritoneal cavity (Supplementary Figure S2) in the 3˚ transplanted mice suggested that the peritoneal cavity may also provide the niche for the fast expansion of leukemic cells. Besides, the presence of leukemic cells in the peritoneal cavity of 1˚ and 3˚ recipients transplanted retro-orbitally indicated the mutual circulation of leukemic cells between the peritoneal cavity and the blood system, justifying the i.p. transplantation. Collectively, these findings validate the use of i.p. injection in serial transplantation of AML.
Figure 2: Generation of murine AML model in 1˚ and 2˚ transplantation. (A) Complete blood count (CBC; 1 x 103 cells/µL blood), WBC, neutrophil (NE), lymphocyte (LY), monocyte (MO), eosinophil (EO), and basophil (BA) profile of 1˚ recipient mice transplanted retro-orbitally with MLL-AF9-transduced bone marrow Lin– cells (5.125 x 106 cells/mouse) by hemavet (n = 3). (B) Flow cytometric analysis of the frequency of AML cells (CD45.1+) in the bone marrow and spleen of 1˚ CD45.2 recipient mice transplanted retro-orbitally with MLL-AF9-transduced bone marrow Lin– (5.125 x 106 cells/mouse) cells (n = 3). (C) Incubation duration of 1˚ AML in recipients transplanted retro-orbitally with MLL-AF9-transduced bone marrow LSK cells (3.16 x 105 cells/mouse isolated from two donors, n = 4) or Lin– cells (3.69 x 106 cells/mouse isolated from two donors, n = 3). Each recipient received cells isolated from two donors. (D) CBC analysis of 1˚ recipient mice transplanted intra-peritoneally with MLL-AF9-transduced bone marrow Lin– cells (5.125 x 106 cells/mouse) by hemavet (n = 4). (E) Flow cytometric analysis of the frequency of AML cells (CD45.1+) in the bone marrow and spleen of 1˚ CD45.2 recipient mice transplanted intra-peritoneally with MLL-AF9-transduced bone marrow Lin– cells (5.125 x 106 cells/mouse, n = 3). (F) Incubation duration of 1˚ AML in recipients transplanted retro-orbitally (n = 3) or intra-peritoneally (n = 3) with MLL-AF9-transduced bone marrow Lin– cells. Each recipient received the same amount of donor cells (5.125 x 106 cells/mouse). (G) CBC analysis of 2˚ recipients transplanted intra-peritoneally with AML cells (8 x 105 cells/mouse) isolated from the spleen of 1˚ i.p. transplanted recipients by hemavet (n = 3). (H) Weights (mg) of the spleen and liver of 2˚ recipient mice transplanted intra-peritoneally with AML cells (8 x 105 cells/mouse) isolated from the spleen of 1˚ i.p. transplanted recipients. The shaded areas represent the normal weight ranges of spleen and liver. Representative image of spleen and liver from 2˚ recipient mice and healthy counterparts. (I) Flow cytometric analysis of the frequency of AML cells (RFP–) in the blood, bone marrow, and spleen of 2˚ RFP+ recipient mice transplanted intra-peritoneally with AML cells (8 x 105 cells/mouse) isolated from the spleen of 1˚ i.p. transplanted recipients (n = 3). (J) Gene expression of KMT2A and 18S presented as a DNA band. RNAs were isolated from the blood, bone marrow, spleen, and liver of 2˚ recipient mice transplanted intra-peritoneally with AML cells (8 x 105 cells/mouse) isolated from the spleen of 1˚ i.p. transplanted recipients. (K) Representative images of histological changes in the spleen (left panel) and liver (right panel) of 2˚ recipients transplanted intra-peritoneally with AML cells (8 x 105 cells/mouse) isolated from the spleen of 1˚ i.p. transplanted recipients and healthy counterparts. (L) Incubation duration of 2˚ AML in recipients transplanted retro-orbitally (4 x 105/mouse, n = 9) or intra-peritoneally (8 x 105/mouse, n = 4) with AML cells isolated from 1˚ r.o. and i.p. transplanted recipients, respectively. The results are mean ± SEM. Please click here to view a larger version of this figure.
Figure 3: 3˚ transplantation of AML cells via i.p. transplantation. (A–E) 3˚ RFP+ or CD45.2 recipient mice. Left: i.p. injection of AML cells (8 x 105 cells/mouse) from the bone marrow of 2˚ recipients. Right: i.p. injection of AML cells (4 x 105 cells/mouse) from the peritoneal cavity (i.p.) of 2˚ recipients (n = 3). (A) CBC analysis of 3˚ recipient mice by hemavet. (B) Weights (mg) of the spleen and liver of 3˚ recipient mice. The shaded areas represent the normal weight ranges of the spleen and liver. (C) Flow cytometric analysis of the frequency of AML cells (left: RFP–; right: CD45.1+) in the blood, bone marrow, and spleen of 3˚ recipient mice. (D) Gene expression of KMT2A and 18S presented as a DNA band. RNAs were isolated from the blood, bone marrow, spleen, and liver of 3˚ recipient mice. (E) Representative images of histological changes in the spleen (left panel) and femur (right panel) of 3˚ recipient mice. (F) Incubation duration of 3˚ AML in recipients transplanted retro-orbitally (4 x 105 cells/mouse, n = 3) or intra-peritoneally (4 x 105 cells/mouse, n = 3) with AML cells isolated from 2˚ r.o. and i.p. transplanted recipients, respectively. The results are mean ± SEM. Please click here to view a larger version of this figure.
Supplementary Figure 1: Flow cytometric analysis of AML cell infiltration in the peritoneal cavity. Frequency of AML cells in the peritoneal lavage of 1˚ recipients transplanted retro-orbitally (1˚ RO, n = 2), 2˚ recipients transplanted intra-peritoneally (2˚ IP, n = 2), and 3˚ recipients transplanted via i.p. injection with bone marrow AML cells (3˚ BM-IP, n = 3) and i.p. AML cells (3˚ IP-IP, n = 3), as well as 3˚ recipients transplanted via r.o. injection of AML splenocytes (3˚ SP-RO, n = 3). The results are mean ± SEM. Please click here to download this File.
Supplementary Figure 2: 3˚ transplantation of AML splenocytes via r.o. injection. (A) CBC analysis of 3˚ recipient mice retro-orbitally transplanted with AML cells (4 x 105 cells/mouse) from the spleen of 2˚ recipient mice. (B) Weights (mg) of spleen and liver of 3˚ retro-orbitally transplanted recipient mice. The shaded areas represent the normal weight ranges of the spleen and liver. (C) Frequency of AML cells (RFP–) in the blood, bone marrow, and spleen of 3˚ retro-orbitally transplanted recipient mice (n = 3). The results are mean ± SEM. Please click here to download this File.
These above-described studies provide supportive evidence that the transplantation of Lin– cells is comparable to LSK cells in the generation of 1˚ murine AML. In addition, the current data also shows that i.p. injection is an efficient and convenient method to establish murine AML compared to intravenous (or r.o.) injection.
In addition to LSK cells, other populations such as granulocyte-monocyte progenitor (GMP), common lymphoid progenitor (CLP), and common myeloid progenitor (CMP) have been reported to be substituted as donor cells in the generation of 1˚ MLL-AF9-induced AML with various incubation durations31,32, although quantitative comparison is lacking to suggest the optimal choice. In the current study, Lin– and LSK isolated from the same donor mice had no apparent difference in the generation of 1˚ MLL-AF9-induced AML, given that transduced myeloid progenitor cells (Sca-1–) were able to initiate AML33. Considering the expense and time required for flow cytometry-based sorting of LSK population, these studies support the use of the Lin– population as the donor cells for 1˚ transplantation. Thus, the steps from 4.19 to 4.23 in this protocol are not necessary but optional, and do not affect the final result.
In terms of the amount of donor mice in 1˚ transplantation, one donor was able to provide ~1.0 to 1.5 x 105 LSK cells, and one recipient transplanted with this amount of LSK cells developed 1˚ AML in about 4 months. However, increasing the number of donors to build up LSK cells up to ~3 x 105 cells per recipient can shorten the incubation duration to ~70 days. Thus, two donors should be enough to rapidly generate 1˚ AML. This principle also applies to Lin– cells, wherein ~1.0 to 2.5 x 106 Lin– cells can be isolated from each donor mouse. However, when donor cell density was increased, the turnaround time of leukemogenesis was considerably shorter. These data should be taken into consideration when deciding the number of donor mice in step 4.1.
Compared to r.o. injection, i.p. injection took about ~20 more days to develop the full-blown 1˚ AML, however this limitation was overcome by increasing the donor cells for i.p. injection. Despite such an apparent difference, both methods showed similar engraftment rate (>80%) in the bone marrow and spleen at the endpoint, indicating the general applicability of i.p. injection in 1˚ transplantation in AML. The relatively long incubation period for 1˚ transplantation, as well as its unpredictable disease progression in terms of inconsistency of latency in recipient mice, is a major limitation for controlled experiments, such as pharmacological interventions and mechanistic studies. Thus, it is recommended to use 1˚ leukemic cells for transplantation in subsequent serial transplantations, typically 2˚ transplantation (Figure 1). Since up to 3 x 108 leukemic cells in spleen (and 6 x 107 cells in bone marrow) can be generated from every single 1˚ recipient at the endpoint, which would be sufficient to conduct 2˚ transplantation on more than 300 mice, it is suggested to transplant as many cells as possible to fewer recipients to shorten the latency and increase the engraftment rate of 1˚ transplantation, which is important for step 4.1 and step 5.2.
Given its consistent and short latency, 2˚ transplantation can be utilized in multiple applications, including in vivo experiments mentioned above. Furthermore, i.p. injection also facilitates the procedure for abundant transplantation for inexperienced technicians. Compared to the r.o. injection route, which generally generates the AML in 3 weeks, i.p. injection method took slightly longer (~4 weeks) with the same number of donor cells to establish a full-blown AML. Even though it is relatively easier and more convenient, increasing the transplanted cell number or performing tertiary transplantation can also accelerate the progression of AML by i.p. injection.
In summary, the major limitation of this technique is the longer incubation time than intravenous injection with the same amount of donor cells in both 1˚ and 2˚ transplantations. However, it can be easily overcome by increasing the cell number to be transplanted. Apart from the obvious advantages, this technique also has some other applications. For example, the ability to use i.p. transplantation methods may also serve to routinely culture these cells without the need for expensive in vitro culture media. To extend the application of this technique in other hematological malignancies, such as lymphoid leukemia and chronic myeloid leukemia, and patient-derived xenografts, further studies need to be done in the future.
The authors have nothing to disclose.
The authors thank Huck Institute's Flow Cytometry Core Facility and the Histopathology Core Facility of the Animal Diagnostic Laboratory, Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, for providing timely technical support. This work was supported by grants from the American Institute for Cancer Research (KSP), Penn State College of Agricultural Sciences, Penn State Cancer Institute, USDA-NIFA project 4771, Accession number 00000005 to K.S.P. and R.F.P.
a-Select competent cells | Bioline | BIO-85027 | |
Ammonium chloride (NH4Cl) | Sigma Aldrich | Cat# A-9434 | |
Ampicillin | Sigma Aldrich | Cat# A0797 | |
Bovine Serum Albumin (BSA), Fraction V—Low-Endotoxin Grade | Gemini bio-products | Cat# 700-102P | |
Ciprofloxacin HCl | GoldBio.com | Cat# C-861-100 | |
DMEM, high glucose, no glutamine | Gibco | Cat# 11960-044 | |
Dulbecco’s Phosphate-Buffered Saline (PBS) | Corning | Cat# 21-031-CV | |
EDTA, Disodium Salt (EDTA-2Na), Dihydrate, Molecular Biology Grade | Calbiochem | Cat# 324503 | |
Fetal Bovine Serum – Premium Select | Atlanta Biologicals | Cat# S11550 | |
Holo-transferrin, bovine | Sigma Aldrich | Cat# T1283 | |
Insulin solution human | Sigma | Cat# I-9278 | |
Iscove's Modified Dulbecco's Medium (IMDM) | Gibco | Cat# 12440-053 | |
L-glutamine 200 mM (100×) solution | HyClone, Gelifesciences | Cat# SH30034.01 | |
LB broth, Lennox | NEOGEN | Cat #: 7290A | |
LB Broth with agar (Miller) | Sigma Aldrich | Cat# L-3147 | |
Mouse anti-mouse CD45.1 (FITC) | Miltenyi Biotec | Cat# 130-124-211 | |
Mouse Interleukin-3 (IL-3) | Gemini bio-products | Cat# 300-324P | |
Mouse Interleukin-6 (IL-6) | Gemini bio-products | Cat# 300-327P | |
Mouse Stem Cell Factor (SCF) | Gemini bio-products | Cat# 300-348P | |
Penicillin-Streptomycin Solution, 100x | Corning | Cat# 30-002-CI | |
Phenix-Eco (pECO) cells | ATCC | CRL-3214 | |
Potassium Bicarbonate (KHCO3), Granular | JT. Baker | Cat# 2940-01 | |
Rat anti-mouse CD117 (c-kit) (APC) | BioLegend | Cat # 105812 | |
Rat anti-mouse Ly-6A/E (Sca-1) (PE-Cy7) | BD Pharmingen | Cat# 558162 | |
Recombinant Murine Flt3-Ligand | Pepro Tech, INC. | Cat# 250-31L | |
RetroNectin Recombinant Human Fibronectin Fragment | TaKaRa | Cat# T100A | |
TransIT-293 Reagent | MirusBio | Cat# MIR 2705 | |
TRI Reagent | Sigma Aldrich | Cat# T9424 | |
Trypan Blue Solution, 0.4% | Gibco | Cat # 15250061 | |
Trypsin-EDTA (0.25%), phenol red | Gibco | Cat# 25200-056 | |
β-Mercaptoethanol (BME) | Sigma Aldrich | Cat# M3148 | |
Commercial Assays | |||
EasySep Mouse Hematopoietic Progenitor Cell Isolation Kit | StemCell technologies | Cat# 19856A | |
High-Capacity cDNA Reverse Transcription Kit | Thermo Fisher | Cat# 4368813 | |
PerfeCTa qPCR SuperMix | Quanta Bio | Cat# 95051-500 | |
Plasmid Maxi Kit (25) | Qiagen | Cat#:12163 | |
Animals | |||
Ai14TdTomato mice | Jackson Laboratory | Strain # 007914 | |
CD45.1 C57BL6/J mice | Jackson Laboratory | Strain # 002014 | |
CD45.2 C57BL6/J mice | Jackson Laboratory | Strain # 000664 | |
Instruments and Softwares | |||
Adobe illustrator | Version 25.2.3 | ||
BD accuri C6 flow cytometer | BD Biosciences | ||
FlowJo 10.8.0 | BD | ||
GeneSys software program | Version 1.5.7.0 | ||
GraphPad Prism version 6 | GraphPad | ||
Hemavet 950FS | Drew Scientific | ||
7300 Real time PCR system | Applied Biosystems | ||
Syngene G:BOX Chemi XR5 Chemiluminescence Fluorescence Imaging | G:Box Chemi |