Can I Centrifuge Samples in Buffer Laemmli Again

• Journal List
• J Vis Exp
• (138); 2018
• PMC6231698

J Vis Exp. 2018; (138): 56855.

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

Xiongwei Cai

¹Division of Experimental Hematology, Cincinnati Children's Hospital Medical Center

²Department of Cell and Developmental Biological science, Abramson Family Cancer Research Institute, Perelman Schoolhouse of Medicine, University of Pennsylvania

^threeDepartment of Obstetrics and Gynecology, Southwest Hospital, Third Military Medical University

Yi Zheng

ⁱSectionalization of Experimental Hematology, Cincinnati Children'southward Hospital Medical Eye

Nancy A. Speck

^twoSection of Cell and Developmental Biology, Abramson Family unit Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania

Abstract

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western blotting protocol was optimized and suitable for the analysis of pocket-size numbers of HSCs (500 - xv,000 cells). Phenotypic HSCs were purified, accurately counted, and direct lysed in Laemmli sample buffer. Lysates containing equal numbers of cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-Folio), and the blot was prepared and processed following standard Western blotting protocols. Using this protocol, 2,000 - 5,000 HSCs can be routinely analyzed, and in some cases data tin be obtained from as few as 500 cells, compared to the 20,000 to forty,000 cells reported in near publications. This protocol should exist generally applicable to other hematopoietic cells, and enables the routine analysis of pocket-size numbers of cells using standard laboratory procedures.

Keywords: Biochemistry, Effect 138, Western blotting, hematopoietic stalk cells, small cell numbers, SDS-Page, beast testing reduce, bone marrow

Introduction

Hematopoietic stalk cells (HSCs) are self-renewing cells that tin can give rise to all blood lineages. They are relatively rare cells in the bone marrow, rendering biochemical analyses difficult. Approaches suitable for analyzing rare cells, such equally menstruation cytometry, take been extremely useful for quantifying relative amounts of jail cell surface markers and intracellular proteins. However, the analysis of intracellular proteins necessitates the apply of cell permeabilization procedures to enable antibody access, and non all cell surface epitopes survive these procedures1,2. In addition, antibodies that discriminate between unlike protein isoforms or cleavage products are not often bachelor for flow cytometry, and therefore investigators notwithstanding rely on Western blots for certain types of analyses.

Western absorb analysis of cell lysates is a routine procedure in nigh laboratories. Cells can be purified under native conditions that preserve the epitopes of cell surface molecules, and cell lysates can later on exist prepared and analyzed. However, the analysis of proteins in rare primary cell populations by Western absorb can require euthanizing large numbers of animals to obtain enough cells. By making small adjustments to several steps, a conventional Western blotting protocol was able to detect proteins in relatively small numbers of HSCs (500 - 15,000, depending on the protein of interest). The adjustments include accurately counting the cells, carefully handling the cell pellet, reducing transfers of cells between tubes to minimize jail cell loss, and lysing a defined number of cells with a concentrated loading buffer containing proteasome and phosphatase inhibitors. Many published reports include Western blots obtained with 20,000 or more than HSCs3,iv,five,half-dozen,vii; this simple process will reduce the number of cells and experimental animals required to produce equivalent data by betwixt iv and forty fold. The protocol is designed to normalize results on a per cell basis, rather than to an internal command. This enables detection of overall reductions in protein levels that can be overlooked if data are normalized to an internal control. The importance of normalizing on a per cell basis was described for the analysis of factor expression data8, and the same principle applies to quantifying proteins by Western blot. This optimized protocol should be useful for anyone needing to analyze small numbers of cells.

Protocol

All procedures must be performed in accordance with institutional animal use and care guidelines. The procedure was adult for the analysis of murine hematopoietic stalk and progenitor cells (HSCs and HPs), simply can be adapted for the analysis of other cell populations.

i. Period Cytometry Isolation of Murine HSCs and HPs

1. Harvest murine bone marrow cells as described in the literature6. NOTE: In data presented in Figure 1 and Figure 2, bone marrow was harvested from C57BL/6J mice.

   

   

2. Perform lineage depletion of bone marrow cells using a mouse lineage depletion kit, following the manufacturer'south instructions.

3. Incubate the cells with antibodies against the cell surface markers of interest as described7. In the experiments shown in Figure one and Figure 2, antibodies to Sca-1, Kit, Flt3, and lineage (Lin) markers Mac1, Gr1, CD3e, B220, and Ter119 are used.

4. Sort 2,000 - 20,000 Lin^- Sca1⁺Kit⁺ Flt3^- cells (HSCs) or Lin^-Sca1^-Kit⁺ cells (HPs) into 1.5 mL Eppendorf tubes containing 0.1 mL of phosphate buffered saline (PBS) with 2% fetal bovine serum (FBS). NOTE: For the information shown in Figure ane and Figure two, cells were sorted using a Flow Cytometer with a lxx µM nozzle. The maximum collection volume is i.four mL.

2. Sample Grooming

NOTE: This step is critical. Procedure the sample very carefully. When removing the supernatant, be very careful non to disturb the prison cell pellet.

1. Centrifuge the 1.v mL tubes containing sorted cells in a swinging bucket rotor at iv °C, 500 ten g, for v min. Remove all but approximately 100 µL of the supernatant from the jail cell pellet very gently using a pipette and a 20 µL pipet tip. Do not disturb the pellet.

   1. If the sample contains fewer than 5,000 cells, and the total book of the sorted sample is less than 0.2 mL, transfer the cells first to low binding 0.2 mL PCR tubes before centrifugation.

   2. If sample contains fewer than 5,000 cells and volume is more than than 0.two mL, spin the cells down with a centrifuge at four °C, 500 x 1000, for 5 min, and remove all but 200 µL of the supernatant. Re-suspend the pelleted cells in the remaining 200 µL of supernatant, and so transfer the cells to the 0.2 mL PCR tubes and spin downwards again. Remove all simply 20 - xxx µL from the pellet after the 2d spin and re-suspend cells.

2. Resuspend the cells in the supernatant left in the tube. If necessary,add additional PBS in 2% FBS/PBS (yous can also use 2% bovine serum albumin (BSA)/PBS, or PBS alone) to obtain a concentration 5 x10^iv to 5 ten 10⁵ cells/mL. NOTE: Use the cell counts nerveless by cytometry to estimate the book used to suspend cells.

3. Utilise ii - 5 µL of the re-suspended cells to decide an accurate cell concentration using an automated prison cell counter (following the manufacturer's instructions) or a hemocytometer9.

4. Transfer a book of re-suspended cells containing the desired number of cells to new 1.5 mL tubes. For the experiment in Effigy ane, 2,000, ane,000, or 500 HSCs or HPs were transferred. The desired number of cells depends on the strength of the bespeak obtained by Western absorb, and is determined empirically. Perform the transfer using a 20 µL or 100 µL Eppendorf pipette tip.

5. Centrifuge the cells in a swinging saucepan rotor at 4 °C, 500 x g, for five min.

6. To generate 100x stock solutions, dissolve proteasome and phosphatase inhibitors in DMSO according to the manufacturer'south instructions.

7. Prepare 2x Laemmli sample buffer by diluting the 4x Laemmli sample buffer provided past the manufacturer with an equivalent amount of distilled water. Add together an appropriate amount of the 100x stock of proteasome and phosphatase inhibitors to obtain a final concentration of 2x inhibitors in 2x Laemmli sample buffer. Note: The 2x Laemmli sample buffer tin can be made in advance, but the proteasome and phosphatase inhibitors should be added immediately before adding the 2x Laemmli sample buffer (plus inhibitors) to the prison cell pellet to lyse the cells, as described in the post-obit step.

8. Advisedly remove a portion of the supernatant from the prison cell pellet, and to the supernatant remaining in the tube with the cell pellet, add an equal book of 2x Laemmli sample buffer plus proteasome and phosphatase inhibitors to achieve a final concentration of 500 cells/µL in 1x Laemmli sample buffer. Note: For example, if you lot transfer 2,000 cells in a full volume of 20 µL to a tube in Footstep 2.5, subsequently centrifuging the cells (Step 2.half dozen) y'all should remove 18 µL of the supernatant from the pellet, and to the 2 µL of supernatant that is remaining add an equal volume (2 µL) of 2x Laemmli sample buffer to achieve a final concentration of 500 cells/µL in 1x Laemmli sample buffer.

9. Resuspend the pellet to generate the lysate every bit soon as possible. At this point, the samples can be immediately electrophoresed through SDS-PAGE gels, or can exist snap frozen in liquid N_ii and stored at -80 °C for future utilise.

iii. Electrophoresis

1. Heat the lysates in a heat block at 95 °C or in boiling h2o for v min.

2. Electrophorese 1 - 40 µL of the lysates (containing from 500 up to xx,000 jail cell equivalents) through SDS-PAGE gels at 100V using standard protocols10. NOTE: The volume of lysate loaded into the gel is determined by the number of cells needed to generate the desirable signal, and will depend on the abundance of the protein of involvement, and the quality of the antibody. The information in Figure 1 were obtained with 2,000, 1,000, and 500 prison cell equivalents of lysate. The width of the well should exist in the range of 3 mm to 1.5 mm. one.5 mm teeth can be cutting from a commercially available comb with wider teeth using pair of scissors.

iv. Transfer and Block

Annotation: Perform a Western blot post-obit standard protocols11. The steps are briefly outlined hither:

1. Pretreat a polyvinylidene difluoride (PVDF) membrane with methanol for 10 s, and so wash the membrane briefly with distilled h2o.

2. Transfer the protein from the SDS-Page gel to the membrane following the instructions in the manual provided by the manufacturer of the transfer apparatus.

3. Following transfer, cake the membrane with two mL of 5% bovine serum albumin (BSA) in PBST (PBS with 0.i% Tween) overnight at 4 °C post-obit standard protocols11.

five. Antibody Labeling

1. Incubate the membrane with antibodies on a shaker overnight at 4 °C using antibody dilutions recommended past the vendor. NOTE: In the Table of Materials, antibodies, which we accept validated for HSCs and HPs and the corresponding antibody dilutions, are shown. Perform the antibody labeling using standard procedures8.

six. Detection

1. Launder the membrane 3 times, 5 min for each wash in PBST at room temperature.

2. Incubate the membrane with 2 mL of enhanced chemiluminescence buffer for 1 min at room temperature. Detect the signals using an imaging system following the manufacturer's instructions, or autoradiography moving picture and picture processor.

Representative Results

Representative results from 500 - 2,000 purified HSCs and HPs are shown in Effigy 1 and Effigy 2. The β-actin point in Figure one tin can be detected from as few as 500 HSCs and HPs purified from the os marrow of one mouse. Note that loading the lysates into 1.5 mm wells produced a much stronger betoken from 500 HPs than loading into 3.0 mm wells. Figure two is a Western blot of EIF4G and the phosphorylation of Rps6 (p-Rps6), both of which are involved in the regulation of protein translation12, in HSCs, and in HPs with and without stimulation by stem jail cell factor (SCF) in vitro.

Figure 1:  Western Blot for β-actin performed with lysates from murine HSCs and HPs. HSCs were sorted from lineage depleted murine bone marrow cells equally Lin^-Sca1⁺Kit⁺ Flt3^- cells, and HPs were Lin^-Sca1^-Kit⁺. Lysates prepared from different numbers of cells were electrophoresed through a 12% SDS-Page gel prepared using mini glass plates with ane mm spacers. The blot was probed with antibody to β-actin. The blot shows the comparative β-actin signals from 500 to 2,000 HPs when the lysates were loaded into 1.five mm wells (lanes 4 - vi) as compared to 3 mm wells (lanes 1 - 2). Lysates from 2,000 to 500 cells were loaded onto lanes vii to 9. One thousand, molecular weight markers. Please click here to view a larger version of this figure.

Figure 2: Western Absorb analysis of freshly isolated HSCs, and HPs stimulated in vitro with the cytokine Stem Jail cell Factor (SCF). HPs were purified by fluorescence activated cell sorting (FACS), centrifuged, resuspended in 2% FBS/PBS, counted, and then split into two tubes. The HPs in the first tube were stimulated with SCF (10 ng/mL) for 5 min at 37 °C (+SCF), and HPs in the 2nd tube were cultured for v min in the absence of SCF (-SCF). The cells were then centrifuged and the jail cell pellet lysed with Laemmli sample buffer. HSCs were purified and directly lysed with Laemmli sample buffer without culturing. The lysates were loaded into i.5 mm wells and electrophoresed through 12% SDS-PAGE gels. The blot was adult with antibodies to elongation initiating cistron 4G (EIF4G), β-actin, and phosphorylated small-scale ribosome subunit S6 (p-Rps6). Please click here to view a larger version of this figure.

Discussion

Western Blotting is a common technique for detecting specific proteins and the activation of signaling pathways in tissues or cells. By introducing modest adjustments to a commonly used procedure, we were able to routinely find 15 different proteins (Tabular array of Materials) in 15,000 HSCs, and in some cases in every bit few as 500 HSCs. The most critical steps in this protocol are: one) accurately counting the cells, 2) minimizing the number of transfers between tubes, and three) lysing the cells directly with Laemmli sample buffer. When lysing the cell pellet with Laemmli sample buffer, nosotros found that rather than removing all of the supernatant from the jail cell pellet and re-suspending it in Laemmli Sample Buffer, if we instead left a small-scale volume of supernatant in the tube and added an equivalent volume of 2x Laemmli Sample Buffer, we could avert cell loss. Centrifuging the cells in a swinging bucket rotor as well decreased the risk of jail cell loss. Reducing the width of the well past trimming the comb teeth improved the sensitivity of detection.

With these simple adjustments to the process, we were able to obtain reproducible results equivalent to those in published papers that had used x - 20 times more cells3,4,v,half dozen,7. Further, the blots can exist stripped for re-blotting following standard procedures13, increasing the corporeality of information that tin exist obtained from a small number of cells. The ability to observe proteins of interest will be limited past the quality of the antibody and the protein abundance. This modified technique should greatly reduce the number of animals necessary to obtain protein data from rare cell populations.

Disclosures

The authors accept no conflict of interests to declare.

Acknowledgments

National Institutes of Wellness grant R01 CA149976 (N.A.Southward) supported this work.

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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6231698/

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