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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
Prepare density centrifugation media mix solution by placing 90 ml of 10x PBS into 264 ml of ultrapure deionized water (3.93x) in a sterile plastic container. Titrate to pH 7.0-7.2 with HCl and filter sterilize through a 0.22 &#181;m filter. Store at 4 &#176;C.
Prepare 70% density centrifugation media by combining 18 ml of density centrifugation media mix solution with 30 ml of density centrifugation media (see materials list for details) in a 50 ml polypropylene centrifuge tube. Store at 4 &#176;C, but bring to RT before use.
Prepare 37% density centrifugation media by combining 9.6 ml of DPBS with 10.4 ml 70% density centrifugation media in a 50 ml centrifuge tube. Store at 4 &#176;C, but bring to RT before use.
1. Isolation of Glioma-infiltrating PBMCs
<ol>
	<li>Using a clean, single-edged razor blade, isolate the area of the brain containing the tumor by making a sagittal cut down the center of the brain to bisect the two hemispheres. Turn the ipsilateral hemisphere medial side down and make two coronal cuts at the levels of the cerebellum and the olfactory bulb to isolate the target tissue containing the tumor implant (Figure 1A). An equivalent portion of the contralateral hemisphere may also be isolated and used as a negative control.</li>
	<li>Place the target tissue into the respectively labeled glass Dounce tissue grinder containing 1 ml of DPBS, push the plunger all the way down, and twist 7 times to initially disrupt the tissue. Lift the plunger to allow the liquid to settle back to the bottom of the tissue grinder. Repeat this step thrice; however, only twist the plunger 4 times during the next two repeats and 3 times during the last repeat to avoid over-triturating the tissue.</li>
	<li>Using a P-1000 micropipette, sequentially apply three 1 ml volumes of ice-cold DPBS along the sides of the plunger to rinse into the tissue grinder.</li>
	<li>Resuspend the triturated brain matter by pipetting up and down and placing it into a labeled 15 ml centrifuge tube on ice. Rinse the sides of the Dounce tissue grinder with 1 additional ml of ice-cold DPBS and add to the same 15 ml centrifuge tube. Keep all tubes containing triturated brain matter on ice until all samples have been processed.</li>
	<li>Once all samples have been processed, spin down the triturated brain matter in the 15 ml centrifuge tubes at 740 x g (max RCF) for 20 min at 4 &#176;C.</li>
	<li>Remove the supernatant with a 10 ml serological pipette and pipette gun and resuspend the pelleted brain matter in 1 ml of the previously prepared collagenase/DNase-I digestive enzymes using a P-1000. Place the 15 ml centrifuge tubes into a test tube rack and place in a 37 &#176;C water bath for 15 min.</li>
	<li>Gently agitate the samples twice throughout the incubation period by flicking the tubes to facilitate tissue disaggregation.</li>
	<li>Add 6 ml of ice-cold DPBS using a 10 ml serological pipette to each tube to dilute the digestive enzymes. Pipette up and down to resuspend, and filter the total volumes through sterile 70 &#181;m nylon mesh filters into new labeled 15 ml centrifuge tubes on ice.</li>
	<li>Spin the brain cell suspension down at 740 x g (max RCF) for 20 min at 4 &#176;C to achieve a single-cell pellet (Figure 1B). After removing the 15 ml tubes from the centrifuge, place them on ice and increase the temperature setting on the centrifuge to about 21 &#176;C in preparation for the next step.</li>
	<li>Fully remove the supernatant from each tube containing brain cells and initially resuspend the pellets in 1 ml of 70% density centrifugation media using a P-1000 micropipette. Then add 4 additional milliliters of 70% density centrifugation media to each tube using a 10 ml serological pipette. Screw the caps on securely and homogenize the cell suspension by gently inverting to tubes several times.</li>
	<li>Remove the 15 ml centrifuge tubes containing brain cells resuspended in 70% density centrifugation media from ice, and place in a test tube rack at RT. One at a time, carefully overlay 2 ml of 37% density centrifugation media solution onto the 5 ml of 70% density centrifugation media with a P-1000 micropipette to form a clean interface between the two density centrifugation media layers (Figure1C).
	<ol>
		<li>Use a fine-tipped marker to indicate the position of the interface so it can be easily identified after centrifugation when the distinction becomes less apparent.</li>
	</ol>
	</li>
	<li>Spin down the 15 ml centrifuge tubes at 740 x g (max RCF) for 20 min at RT with no break to avoid disrupting the interface.</li>
	<li>After centrifugation, collect the PBMCs that have accumulated at the interface between the two density centrifugation media layers (Figure 1D) by introducing a P-200 micropipette into the tube along its side, carefully bypassing the lipid layer.
	<ol>
		<li>Once at the level of the PBMC band, slowly extract 200 &#181;l from the surface of the 70% density centrifugation media layer and place it into a respectively labeled polypropylene FACS tube on ice. Repeat once for a total volume of 400 &#181;l per sample.</li>
	</ol>
	</li>
	<li>Add 3 ml of flow buffer to each FACS tube containing 400 &#181;l of PBMCs to sufficiently dilute the density centrifugation media, then spin the cells down at 660 x g (max RCF) for 20 min at 4 &#176;C.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modification of Eagles Medium</td>
			<td>Gibco</td>
			<td>12430-054</td>
			<td>With 4.5g/L D-glucose, L-glutamine, 25mM HEPES and phenol red</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-buffered Saline</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>Without calcium chloride or Magnesium chloride</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Omega Scientific Inc.</td>
			<td>FB-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>10,000U/ml Penicillin; 10,000&#956;g/ml Streptomycin</td>
		</tr>
		<tr>
			<td>0.6ml conical polypropylenemi microtubes</td>
			<td>Genesee Scientific</td>
			<td>22-272</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyurethane ice bucket</td>
			<td>Fisherbrand</td>
			<td>02-591-45</td>
			<td>Capacity: 0.152 oz. (4.5L)</td>
		</tr>
		<tr>
			<td>Collagenase (Type I-S)</td>
			<td>Sigma Aldrich</td>
			<td>C1639</td>
			<td>From Clostridium histolyticum</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I</td>
			<td>Worthington Biochemical Corp.</td>
			<td>LS002007</td>
			<td>&#62;2,000 Kunitz units per mg dry weight</td>
		</tr>
		<tr>
			<td>Large dissection scissors</td>
			<td>Ted Pella Inc.</td>
			<td>1316</td>
			<td></td>
		</tr>
		<tr>
			<td>Small dissection scissors</td>
			<td>FST</td>
			<td>14094-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt end forceps</td>
			<td>Ted Pella Inc.</td>
			<td>13250</td>
			<td></td>
		</tr>
		<tr>
			<td>7ml glass Dounce tissue grinder</td>
			<td>Kontes</td>
			<td>KT885300-0007</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll Density Centrifugation Media</td>
			<td>GE Healthcare</td>
			<td>GE17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL serological pipettes</td>
			<td>Genesee Scientific</td>
			<td>12-104</td>
			<td>Single use; sterile</td>
		</tr>
		<tr>
			<td>70&#956;m sterile nylon mesh cell strainers</td>
			<td>Fisherbrand</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr>
			<td>15ml polypropylene centrifuge tubes</td>
			<td>Genesee Scientific</td>
			<td>21-103</td>
			<td>Conical bottom</td>
		</tr>
		<tr>
			<td>50ml polypropylene centrifuge tubes</td>
			<td>Genesee Scientific</td>
			<td>21-108</td>
			<td>Conical bottom</td>
		</tr>
		<tr>
			<td>12x75mm round-bottom polypropylene FACS tubes</td>
			<td>Fisherbrand</td>
			<td>14-956-1B</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating tumor-infiltrating immune cells from a murine brain

Source: Baker, G. J., et al., Lowenstein, P. R. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53676">Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells.</a> J. Vis. Exp. (2015)
The video demonstrates the isolation of tumor-infiltrating peripheral blood mononuclear cells from a mouse brain using mechanical dissociation and enzymatic digestion. This is followed by density gradient centrifugation to collect the immune cells for further analysis.

<img alt="Figure 1" src="/files/ftp_upload/22387/22387_Figure1.jpg" /> 
Figure 1: Isolation of Glioma-infiltrating PBMCs. (A) Strategy for the dissection of mouse brain tissue containing early-stage orthotopic glioma implants. The top panel demonstrates the sagittal bisection of the ipsilateral hemisphere away from the contralateral hemisphere. The bottom panel demonstrates the two additional coronal cuts necessary to isolate the target tissue containing the tumor implant (highlighted in purple). (B) Single-cell brain tissue pellet (white arrow) after enzymatic digestion, filtration through a 70 &#956;m nylon mesh, and centrifugation. (C) Properly poured density centrifugation media gradient prior to centrifugation. The white arrow indicates the clean interface formed between the two density centrifugation media layers. (D) Density centrifugation media gradient after centrifugation demonstrating the lipid layer that forms at the top of the 37% density centrifugation media layer white arrow) and the PBMC band (outlined by the dashed black lines) at the interface between the two layers.

Isolating Tumor-Infiltrating Immune Cells from a Murine Brain

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
Prepare density centrifugation media ...

Take a perfused mouse brain containing a tumor.
The perfusion step removes circulating immune cells but retains the tumor-infiltrated cells.
Isolate the tissue containing the tumor.
Now, mechanically dissociate the tissue.
The mechanical force loosens the tissue, releasing the cells.
Transfer the dissociated tissue to a tube.
Centrifuge and discard the supernatant containing debris.
Resuspend the tissue in a digestion solution containing collagenase and DNase.
Collagenase degrades the extracellular matrix, releasing the cells. DNase degrades contaminating DNA.
Add a buffer to stop the enzymatic activity.
Filter the suspension to remove cell aggregates and obtain a single-cell suspension.
Centrifuge and discard the supernatant.
Resuspend cells in a high-density gradient medium. Now, overlay with a lower-density gradient medium, forming a separation gradient with a clear interface.
Centrifuge to separate layers. Immune cells gather at the interface between gradient mediums while cell debris floats atop.
Collect the immune cells for further analysis.

isolating-tumor-infiltrating-immune-cells-from-a-murine-brain

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Co-culture and Interaction Studies

53 조회수. 출처: Baker, G. J., et al., Lowenstein, P. R. 신경교종 침투 말초 혈액 단핵 세포의 분리 및 유세포 분석. J. Vis. 특급. (2015년) 이 비디오는 기계적 해리와 효소 분해를 사용하여 쥐의 뇌에서 종양에 침투하는 말초 혈액 단핵 세포를 분리하는 방법을 보여줍니다. 그 다음에는 추가 분석을 위해 면역 세포를 수집하기 위해 밀도 구배 원심분리가 수행됩니다. 

비디오: 쥐의 뇌에서 종양 침투 면역 세포 분리 - 개념

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

인간의 흑색 종 종양 침윤 림프구에서 유도 만능 줄기 세포의 생성

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

연구

JoVE Journal

발생학

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

마우스에서 종양 침윤성 CD8+ T 세포의 역학을 평가하기 위한 종양 이식

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

암 연구학

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

효소 다이제스트 및 그라데이션 분리를 사용하여 마우스 피부에서 침투 백혈구의 분리

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Isolating Tumor-Infiltrating Immune Cells from a Murine Brain

내레이션 대본

Isolating Tumor-Infiltrating Immune Cells from a Murine Brain