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All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Intraperitoneal Injection of Cells
NOTE: In our experiments, mice are handled in a laminar airflow or biosafety cabinet within our barrier facility in order to limit the risk of pathogen exposure. In order to demonstrate this technique clearly, the procedures in the accompanying video were conducted in a laboratory approved for animal work.&#160;This particular technique does not require the animals to be under anesthesia. Under approved protocols, perform this technique on live animals. Use appropriate strains of mice for this study. For example: use immunocompromised, athymic nude mice for the study of human ovarian cancer cell (SKOV3ip.1 and HeyA8) colonization; use immunocompetent C57BL/6 mice for study of mouse ovarian cancer cell (ID8) colonization.
<ol>
	<li>Load 500 &#181;l of single cell suspension of ovarian cancer cells into a syringe and put in the sterile capped 25 G needle. This reduces cell shearing prior to injection.</li>
	<li>Pick the mouse up by the scruff of the neck and hold the tail using the palm and forefinger and fix the left hind leg between the ring and little finger (when the mouse is restrained with the left hand). 
	NOTE: To avoid traumatizing abdominal organs, restrain the mouse well so that it cannot move during the injection.</li>
	<li>Imagine a line across the abdomen just above the knees, and locate a point on the animal&#39;s right side and close to the midline. The point of entry is cranial to and slightly medial of the last nipple. 
	NOTE: Inserting the needle on the mouse&#39;s right side avoids the cecum and reduces the risk of puncturing the intestines.</li>
	<li>Insert the needle at the lower lateral region of the mice&#39;s abdomen to a depth of approximately 0.5 cm. Pull back on the plunger to confirm that the needle has not penetrated a blood vessel or other peritoneal organs.
	<ol>
		<li>If any fluid is aspirated discard the syringe (and sample). A greenish-brown or yellow aspirate indicates needle penetration into the intestines or bladder, respectively.</li>
	</ol>
	</li>
	<li>Inject the sample using slow, steady pressure. Withdraw the needle and return the mouse to its cage. Do not recap the syringe before disposal in a sharp container.</li>
	<li>Sacrifice mice via CO2&#160;asphyxiation and vital organ removal at a specific time point post injection.</li>
</ol>

<table><tbody><tr><td>DMEM</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>PBS</td><td>Corning</td><td>21-040-CV</td><td>Without calcium and Magnesium</td></tr><tr><td>26 gauge needle</td><td>BD</td><td>329652</td><td></td></tr></tbody></table>

intraperitoneal injection of cells: a method of delivering cancer cells into the peritoneal adipose tissue of murine model

Source: Krishnan, V. et al. <a href="https://www.jove.com/t/52721">Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose.</a> J. Vis. Exp. (2015)
In this video, we perform an intraperitoneal injection of ovarian cancer cell suspension in a mouse model to study the colonization of ovarian cancer cells in peritoneal adipose tissues and their subsequent metastasis.&#160;

Intraperitoneal Injection of Cells: A Method of Delivering Cancer Cells into the Peritoneal Adipose Tissue of Murine Model

Source: Krishnan, V. et al. Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose. J. Vis. Exp. 
...

The peritoneal cavity is the space between the abdominal wall and the lining of the internal organs. For intraperitoneal injection, begin by restraining a live mouse by its neck region such that the mouse&#8217;s head tilts downward with its ventral abdomen exposed.
Restraining the mouse restricts its movement and shifts the abdominal organs towards the diaphragm, preventing accidental damage to the organs during needle insertion.
Adjacent to the midline across the mouse&#8217;s abdomen, in the lower right quadrant, locate a point for needle insertion. This site prevents the risk of puncturing the caecum.
Next, take a syringe containing a single cell suspension of ovarian cancer cells. Insert the needle at the chosen point to a depth of approximately half a centimeter to penetrate just the outer abdominal wall. Finally, inject the cancer cell suspension slowly. This ensures efficient distribution of cells within the peritoneal cavity.
Return the mouse to its cage and monitor it for tumor growth. The tissues within the peritoneal cavity may release specific chemotactic molecules that attract the cancer cells leading to their migration and colonization, eventually contributing to tumor development.

intraperitoneal-injection-cells-method-delivering-cancer-cells-into

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Cancer Research

Gynecologic Cancer

Animal model studies

4.8K 조회수. 출처: Krishnan, V. et al. 복막 지방에서 은하수 반점 구조의 난소암 전이성 집락화를 연구하기 위한 접근 방식. J. Vis. 특급. (2015년) 이 비디오에서는 복막 지방 조직에서 난소암 세포의 집락화와 그에 따른 전이를 연구하기 위해 마우스 모델에서 난소암 세포 현탁액의 복강 내 주사를 수행합니다. 

비디오: 세포의 복강 내 주입: 쥐 모델의 복막 지방 조직에 암세포를 전달하는 방법 - 개념

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such as Chronic Wasting Disease (CWD) of cervids and scrapie in sheep. The Prion hypothesis asserts that this abnormal conformer constitutes most or all of the infectious prion. The role of the immune system in early events in peripheral prion pathogenesis has been convincingly demonstrated for CWD and scrapie 1-3. Transgenic and pharmacologic studies in mice revealed an important role of the Complement system in retaining and replicating prions early after infection 4-6. In vitro and in vivo studies have also observed prion retention by dendritic cells 7-10, although their role in trafficking remains unclear 11-16. Macrophages have similarly been implicated in early prion pathogenesis, but these studies have focused on events occurring weeks after infection 3,11,17. These prior studies also suffer from the problem of differentiating between endogenous PrPC and infectious prions. Here we describe a semiquantitative, unbiased approach for assessing prion uptake and trafficking from the inoculation site by immune cells recruited there. Aggregated prion rods were purified from infected brain homogenate by detergent solubilization of non-aggregated proteins and ultracentrifugation through a sucrose cushion. Polyacrylamide gel electrophoresis, coomassie blue staining and western blotting confirmed recovery of highly enriched prion rods in the pelleted fraction. Prion rods were fluorochrome-labeled then injected intraperitoneally into mice. Two hours later immune cells from peritoneal lavage fluid, spleen and mediastinal and mesenteric lymph nodes were assayed for prion rod retention and cell subsets identified by multicolor flow cytometry using markers for monocytes, neutrophils, dendritic cells, macrophages and B and T cells. This assay allows for the first time direct monitoring of immune cells acquiring and trafficking prions in vivo within hours after infection. This assay also clearly differentiates infectious, aggregated prions from PrPC normally expressed on host cells, which can be difficult and lead to data interpretation problems in other assay systems. This protocol can be adapted to other inoculation routes (oral, intravenous, intranervous and subcutaneous, e.g.) and antigens (conjugated beads, bacterial, viral and parasitic pathogens and proteins, egg) as well.

Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.

Intraperitoneal 감염 후 면역 세포의 인신 매매 형광등 프리온 로즈 시간을 모니터링

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

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JoVE Journal

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Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

성인 Zebrafish에 Intraperitoneal 사출

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

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Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats

Cerebral microhemorrhages (CMHs) are common in aged patients and are correlated to various neuropsychiatric disorders. The etiology of CMHs is complex, and neuroinflammation is often observed as a co-occurrence. Here, we describe a sub-acute CMHs rat model induced by lipopolysaccharide (LPS) injection, as well as a method for detecting CMHs. Systemic LPS injection is relatively simple, economical, and cost-effective. One major advantage of LPS injection is its stability to induce inflammation. CMHs caused by LPS injection could be detected by gross observation, hematoxylin and eosin (HE) staining, Perl&#39;s Prussian staining, Evans blue (EB) double-labeling, and magnetic resonance imaging-susceptibility weighted imaging (MRI-SWI) technology. Finally, other methods of developing CMHs animal models, including their advantages and/or disadvantages, are also discussed in this report.

We present a protocol to induce and detect CMHs caused by LPS injection in Sprague-Dawley rats, which may be utilized in future research investigations on the pathogenesis of CMHs.

아 급성 대뇌 Microhemorrhages 쥐에서 Lipopolysaccharide 주입에 의해 유도 된

Cerebral microhemorrhages (CMHs) are common in aged patients and are correlated to various neuropsychiatric disorders. The etiology of CMHs is complex, ...

Intraperitoneal Injection of Cells: A Method of Delivering Cancer Cells into the Peritoneal Adipose Tissue of Murine Model

내레이션 대본

Intraperitoneal Injection of Cells: A Method of Delivering Cancer Cells into the Peritoneal Adipose Tissue of Murine Model